Treatment of rumen acidosis with α-amylase inhibitors

ABSTRACT

The invention described herein relates to: the use of an effective inhibitor of a bacterial α-amylase and/or α-glucosidase in the manufacture of a composition for the treatment of rumen acidosis; a method of treatment of rumen acidosis which comprises administration of an effective amount of an effective inhibitor of a bacterial α-amylase and/or α-glucosidase to a ruminant; a formulation suitable for the treatment of rumen acidosis in an animal which comprises an effective inhibitor of a bacterial α-amylase and/or α-glucosidase in admixture with a suitable excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical/veterinary/farming practice; screening methods useful in the identification of a suitable inhibitor of a bacterial α-amylase and/or α-glucosidase for the treatment of acidosis in a ruminant; a process for improving ruminant milk quality and/or quantity which comprises treatment of a ruminant with an effective amount of an inhibitor of bacterial α-amylase and/or α-glucosidase; a compound of the formula I: 
                 
 
or veterinarily acceptable salt, solvate (including hydrate) or prodrug thereof; and processes to make an effective inhibitor of a bacterial α-amylase and/or α-glucosidase useful for the treatment of acidosis in a ruminant.

This application is a continuation of U.S. patent application Ser. No.09/864,515, filed on May 24, 2001, now abandoned, which claims priorityof U.K. Patent Application No. 0012793.6, filed on May 24, 2000, U.K.Patent Application No. 0012760.5, filed on May 24, 2000, U.K. PatentApplication No. 0017495.3, filed on Jul. 17, 2000, and the benefit ofU.S. patent application Ser. No. 60/218,494, filed on Jul. 14, 2000,U.S. patent application Ser. No. 60/218,326, filed on Jul. 14, 2000, andU.S. patent application Ser. No. 60/225,156, filed on Aug. 14, 2000.

The invention described herein relates to the treatment of rumenacidosis, especially chronic acidosis in ruminants, and relatedconditions.

Rumen acidosis is a well-documented metabolic disease of ruminantscaused by over-consumption of readily fermentable carbohydrates, andproblems associated with the condition have been known for many years:see Nordlund et al. 1995, Nagaraja et al. 1998, Owens et al. 1998 andDirksen 1969. Acidosis can be divided into two forms: acute and chronic.We define acute acidosis as a rumen pH between pH 4.0 and 5.0 withelevated ruminal lactate, and chronic acidosis as a rumen pH between 5.0and 5.5 with normal levels of lactate of up to 5 mM. The literature alsorefers to subacute acidosis, which has rumen pH values below 5.0 but insome cases is associated with high lactate levels and in others is not.We categorise the former case as mild acute acidosis, and the latter aschronic acidosis.

The main cause of acidosis is the consumption of a diet with a highcontent of readily fermentable carbohydrate and/or which is low inroughage. Chronic acidosis can occur when animals eat large quantitiesof readily fermentable diets and may occur at any stage in production,or indeed throughout the time that they are on the high concentratediets. Acute acidosis can occur when a large increase in the amount ofconcentrate in the diet takes place, for example after calving or ontransfer to the feedlot. However it can also occur following adisruption in normal feed intake patterns such as accidentalpresentation of excess feed or a fasting period followed by overeating.Reduced rumen pH can also be caused by a decrease in the proportion ofcrude fibre in the diet. The aetiology of acidosis is therefore based onthe absolute intake of excessive quantities of carbohydrate and/or anunfavourable proportion of basic foodstuffs in the ration. The type ofgrain (high moisture corn is more acidosis-inducing than dry-rolled cornor sorghum) and the type of processing (steam flaked grain isparticularly digestible) along with type and amount of roughage isimportant. Grains such as barley, wheat and high-moisture corn that havefast rates of ruminal starch digestion generally cause the mostproblems. For example barley, wheat flour, oats and steam flaked cornall have ruminal starch availability greater than 85%. Guidelines fordiets for dairy cattle producing more than 35-40 kg of milk suggestneutral detergent fibre of 25-30% of the diet, with 75% of that fromforage, non-structural carbohydrate levels of 35-40% and starch of30-40% (Nocek 1997).

Acute acidosis is characterised by a precipitous decrease in ruminal pHwith a high concentration of ruminal lactic acid (50-100 mM). Theruminal microbial population undergoes a significant shift, with anincrease in gram-positive lactic-acid producing bacteria, specificallyStreptococcus bovis and Lactobacillus species. The falling pH leads tothe death of gram-negative bacteria and the reduction or completedisappearance of ciliated protozoa. The shift in the fermentationpattern to lactate production is associated with decreased volatilefatty acid (VFA) production. Systemic changes include decreased blood pHand bicarbonate and increased blood D and L-lactate. Acute acidosis cancause significant impairment of physiological functions such as ruminalstasis and dehydration, eventually leading to coma and death. Even ifthe animal survives, it may never completely recover.

Chronic acidosis has much more subtle clinical signs. The animals remainalert and consume feed, but may look ‘off-colour’. The fall in rumen pHto below 5.5 is due to a general increase in fermentation within therumen leading to greater production of VFAs. The increase of VFAs in therumen is very highly correlated with increases in the blood, but bloodpH does not change significantly. Total ciliated ruminal protozoadecline due to the falling pH, with species differences in rate, but donot disappear entirely. Total viable bacterial counts increase overtime, including increased amylolytic bacteria. However the overwhelmingrise in S. bovis and Lactobacillus species seen in acute acidosis doesnot occur. While the rate of lactate production rises transiently afterfeeding the lactate is utilised immediately in production of VFAs, anddoes not accumulate in the rumen. Specifically the symptoms of chronicacidosis are a fall in ruminal pH to 5.0-5.5 without significant lacticacid accumulation.

Summary of symptoms of acute and chronic acidosis Normal Chronicacidosis Acute acidosis Rumen pH >6.0 5.5-5.0 <5.0 VFAs ˜100 mM up to200 mM reduced lactate concentration up to 5 mM up to 5 mM >50 mMglucose negligible negligible >10 mM Protozoa much reduced dead Bacteriaincreased increased S. bovis and Lactobacillus sp.

Rumen acidosis is associated with many secondary conditions that canhave a significant impact on livestock animal performance, i.e.reduction in the feed conversion to meat and/or milk. Milk quality canalso suffer in association with acidosis. Irreversible damage to theruminal epithelium occurs at a rumen pH below 5.5, causinghyperkeratosis, papillary clumping and rumenitis of the ruminalepithelium. The animals have reduced appetite and performance due toimpaired nutrient absorption, resulting in reduced weight gain in beefcattle and decreased milk yield and quality in dairy cattle. Othereffects are laminitis, intermittent diarrhoea, poor appetite and cyclicfeed intake, a high herd cull rate for poorly defined health problems,poor body condition and abscesses without obvious causes. Chroniclaminitis is one of the most consistent clinical signs, with ridges inthe dorsal hoof wall, sole ulceration, white line lesions, solehaemorrhages and misshapen hooves. On average, farmers report that 25%of animals in UK dairy herds are lame, and the true incidence of chroniclaminitis is likely to be higher as it does not always producedetectable lameness. Liver abscesses are known to be linked withacidosis, and in most feedlots the incidence of liver abscesses averagesfrom 12% to 32% of slaughtered cattle, and is a major cause of livercondemnation. Liver abscesses are not necessarily diagnosed while theanimal is alive, but have a deleterious effect on their performance andgeneral health. Animals may also have depressed immune function, a highincidence of respiratory diseases and reduced fertility rates. Mostdairy herds with a chronic acidosis problem have an annual herd turnoverrate of greater than 45%, or an annual cull rate greater than 31%. Thereasons for culling are usually poorly defined. (Nocek 1997, Nordlund1995, Nagaraja 1998, Stock and Britten 1998, AnimalPharm 1999, Kay 1969,McManus 1977).

Another problem which can be seen with high-yielding dairy cows fed witha high carbohydrate and/or low roughage diet is the acidosis-related“low milk fat syndrome”. As the pH in the rumen falls, the pattern offermentation shifts towards producing more propionate and less acetateand butyrate. As approximately half of milk fat is produced from acetateand butyrate, this results in a drop in the milk fat content. (A TChamberlain & J M Wilkinson, Feeding the Dairy Cow, ChalcombePublications, UK, 1996).

Rumen acidosis and related problems are estimated to cost the livestockindustry more than $1 billion per annum due to lost performance.

Recommended treatments for acute acidosis include administration of amixture of sodium bicarbonate, formaldehyde, magnesium oxide andcharcoal to kill rapidly dividing bacteria. (NebGuide G91-1047-A).Buffers are widely used (Horn 1979, Kennelly 1999), but do not seemefficacious enough to satisfy the livestock industry. Palatability ofmost buffers is low, and requires careful management to avoid reducedfeed intake. Ionophore antibiotics such as monensin, lasalocid andsalinomycin are generally effective against gram-positive bacteria,including the major ruminal lactate-producing bacteria, S. bovis andLactobacillus species (Burrin and Britton 1986, Coe 1999, Nagaraja1985). They are therefore effective at preventing acute acidosis ontransfer to high concentrate diets when cattle first reach the feedlotor following calving. They also act to reduce total VFA production incattle with chronic acidosis, and therefore stabilise rumen pH. Howeverionophores also decrease food intake. Other antibiotic classes have alsobeen shown to prevent or ameliorate acute acidosis, includingvirginiamycin in sheep (Thorniley et al 1998), and thesulphur-containing peptide antibiotic thiopeptin, which is particularlyeffective against S. bovis (Armstrong 1984). However, sustained use ofantibiotic feed additives is no longer seen as an appropriate managementtool (for review see: The use of drugs in food animals: benefits andrisks, 1999). Probiotic control has been demonstrated with a number ofspecies, including Selenomonas ruminantium subsp. lactolytica strainJDB201 (Wiryawan et al 1995), the lactate utlilizer Megasphaeraelsedenii (Das, Kung and Hession 1995), and in more general terms patentWO 96/17525. The latter also claims enzymes that increase degradation ofstarch or fibre. Other proposed, but not commercialised, treatmentsinclude use of bacteriocins (Teather and Forster 1998), and theeconomically unviable manipulation of ruminal fermentation with organicacids (Martin, 1988, Martin et al. 1999).

-   -   Armstrong, D. G. Antibiotics as feed additives for ruminant        livestock in ‘Antimicrobials and Agriculture The proceedings of        the 4^(th) International symposium on antibiotics in        agriculture: benefits and malefits’, 1984 ed. Woodbine M.        Butterworths ISBN 0 408 11155 0    -   Das, N. K. Ruminant feed additive Patent application US        76-748210 761207    -   Kennelly, J. J., Robinson, B. and Khorasani, G. R. Influence of        carbohydrate source and buffer on rumen fermentation        characteristics, milk yield, and milk composition in        early-lactation Holstein cows Journal of Dairy Science 1999 82:        2486-2496    -   Kung, L. and Hession, A. O. Preventing in vitro lactate        accumulation in ruminal fermentations by inoculation with        Megasphaera elsedenii Journal of Animal Science 1995 73: 250-256    -   Martin, S. A. Manipulation of ruminal fermentation with organic        acids: a review Journal of Animal Science 1998 76: 3123-3132    -   Martin, S. A., Streeter, M. N., Nisbet, D. J., Hill, G. M. and        Williams, S. E. Effects of DL-Malate on ruminal metabolism and        performance of cattle fed a high-concentrate diet Journal of        Animal Science 1999 77: 1008-1015    -   Teather, R. M. and Forster, R. J. Manipulating the rumen        microflora with bacteriocins to improve ruminant production        Canadian Journal of Animal Science 1998 78 (Supplement): 57-69.    -   The use of drugs in food animals: benefits and risks CABI        publishing 1999 ISBN 0 85199 371 0    -   Thorniley, G. R., Rowe, J. B., Cowcher, P. C., Boyce, M. D. A        single drench of virginiamycin to increase safety of feeding        grain to sheep Australian Journal of Agricultural Research 1998        49 (5): 899-906    -   Wiryawan, K. G. and Brooker, J. D. Probiotic control of lactate        accumulation in acutely grain-fed sheep Australian Journal of        Agricultural Research 1995 46 (8):1555-68    -   Kay, M., Fell, B. F. and Boyne, R. The relationship between the        acidity of the rumen contents and rumenitis in calves fed on        barley Research in Veterinary Science 1969 10 181-187    -   McManus, W. R., Lee, G. J. and Robinson, V. N. E. Microlesions        on rumen papillae of sheep fed diets of wheat grain Research in        Veterinary Science 1977 22: 135-137

Further References

-   1. Lameness costs UK dairy herds, says NMR AnimalPharm 417 Mar.    26^(th) 1999 p6-   2. Burring, D. G. and Britton, R. A. Response to monensin in cattle    during subacute acidosis Journal of Animal Science 1986 63:888-893-   3. Coe, M. L., Nagaraja, T. G., Sun, Y. D., Wallace, N. Towne, E.    G., Kemp, K. E. and Hutcheson, J. P. Effect of Virginiamycin on    ruminal fermentation in cattle during adaptation to a high    concentrate diet and during an induced acidosis Journal of Animal    Science 1999 77:2259-2268-   4. Dirksen, G. Acidosis in Physiology of Digestion and Metabolism in    the Ruminant: Proceedings of the Third International Symposium,    Cambridge, England: August 1969 Ed. A. T. Phillipson, Oriel Press    IBSN 0 85362 053 9-   5. Nagaraja, T. G. Galyean, M. L. and Cole, N. A. Nutrition and    Disease Veterinary Clinics of North America: Food Animal Practice    1998 14 (2) 257-277-   6. Nagaraja, T. G., Avery, T. B., Galitzer, S. J. and Harmon, D. L.    Effect of ionophore antibiotics on experimentially induced lactic    acidosis in cattle American Journal of Veterinary Research 1985    46 (12) 2444-2452-   7. Nocek, J. E. Bovine acidosis: Implications on laminitis Journal    of Dairy Science 1997 80:1005-1028-   8. Nordlund, K. V. Garrett, E. F. Oetzel, G. R Herd-based    rumenocentesis: a clinical approach to the diagnosis of subacute    rumen acidosis. Compendium on Continuing Education for the    Practicing Veterinarian. 1995. 17: 8, Supplement, S48-S56-   9. Owens, F. N. Secrist, D. S. Hill, W. J. and Gill D. R. Acidosis    in cattle: a review Journal of Animal Science 1998 76:275-286-   10. University of Nebraska, Lincoln NebGuide G91-1047-A    http:/www.inar.unl.edu/pubs/AnimalDisease/g1047. htm

There is a general need for a safe effective treatment for rumenacidosis;

especially chronic and/or acute rumen acidosis;

especially in ruminants such as cattle and sheep;

especially in lactating ruminants such as cattle and sheep;

which can preferably be administered easily, such as with food or drink;

which preferably is non-antimicrobial;

preferably which is palatable to the animal;

preferably which is active only in the rumen and has no systemiceffects;

which preferably does not present any residues in meat and/or milk, and

which preferably does not require a withholding period;

which is preferably non-toxic to animal and feed handlers (manufacturerand farmer);

and/or which preferably can stabilise the rumen fermentation, thuspreventing excessive reductions in pH and maintaining VFA proportionssuch that milk fat production is not adversely affected.

We have discovered that certain inhibitors of bacterial α-amylase and/orα-glucosidase can be used to reduce ruminal pH in an effective way whichshould be useful in the treatment of both chronic and acute acidosis andrelated conditions.

By “inhibitor” herein is meant individual agents and mixtures of agentswhich have inhibitory activity, including fermentation broth productsmentioned below.

One aspect of the invention is the use of an effective inhibitor ofbacterial α-amylase and/or α-glucosidase in the manufacture of acomposition for the treatment of acidosis. Of particular interest areinhibitors of amylases/glucosidases present in ruminal bacteria, such asthose mentioned hereinafter.

A further aspect of the invention is a method of treatment of acidosiswhich comprises administration of an effective amount of an inhibitor ofbacterial α-amylase and/or α-glucosidase to an animal.

A further aspect of the invention is a formulation suitable for thetreatment of acidosis in an animal which comprises an inhibitor ofbacterial α-amylase and/or α-glucosidase.

Further aspects of the invention are as defined in the claims.

Preferably the inhibitor of bacterial α-amylase and/or α-glucosidase hasan IC₅₀ of 10⁻³M or less, more preferably 10⁻⁴M or less, yet morepreferably 10⁻⁵M or less, in the rumen amylase and glucosidase screensdescribed herein.

Preferably the amylase and/or α-glucosidase inhibitor has lowantimicrobial activity, more preferably with a MIC value of more than 50μg/ml in the tests described herein, yet more preferably more than 100μg/ml.

A preferred group of α-amylase and/or α-glucosidase inhibitors includethe substances disclosed below and simple analogues thereof, includingin the Examples below, which are found to be effective in the screensmentioned below (NB All references mentioned herein are herebyincorporated in their entirety):

defined as “acarbose and higher homologues.”*

Compound disclosed specifically in DE-2347782. The following arehomologues of acarbose:

These specific compounds are disclosed in GB-1,482,543, wherein:

X M N GB-1,482,543 ref OH 0 0 Component II OH 0 1 Component III OH 0 2Component IV OH 0 3 Component V OH 0 4 Component VI OH 0 5 Component VIIOH 0 6 Component VIII

These specific compounds are disclosed in Agric. Biol. Chem., 46 (7),1941-1945, 1982, wherein:

X M N Agr. Biol. Chem ref OH 0 0 Component 1 OH 0 1 Component 2 OH 0 2Component 3 OH 1 0 Component 4 OH 1 1 Component 5 OH 1 2 Component 6

The following semi-synthetic acarbose analogues, generically andspecifically disclosed in U.S. Pat. No. 4,175,123

wherein M=0 to 8, and the sum of M+N is 0 to 7; X is OR, SH, SR, NH₂,NHR, or NRR¹, where R is alkyl, alkenyl, cycloalkyl, aralkyl, aryl orheterocyclyl wherein:

alkyl is preferably straight-chain or branched alkyl with 1 to 30,especially 1 to 18, carbon atoms (e.g. methyl, ethyl, n-propyl,1-propyl, n-butyl, t-butyl, n-hexyl, n-octyl, octyl-2, dodecyl, lauryl,cetyl and stearyl), wherein the alkyl radicals R can carry one or more,preferably 1 to 5, identical or different substituents (e.g., hydroxyl,or alkoxy, with preferably 1 to 4 carbon atoms, methoxy and ethoxy;amino or monoalkylamino and dialkylamino, with preferably 1 to 4 carbonatoms per alkyl radical, monomethylamino, monoethylamino, dimethylamino,and diethylamino; mercapto or alkylthio, with preferably 1 to 4 carbonatoms, methylthio and ethylthio; halogen (preferably fluorine, chlorineand bromine); alkylcarbonyl, with preferably 1 to 4 carbon atoms in thealkyl radical; and carboxyl, nitro, cyano, the aldehyde group and thesulphonic acid group;

alkenyl is preferably straight-chain or branched alkenyl with 2 to 6carbon atoms, with optional substituents (e.g. hydroxyl, alkoxy with 1to 4 carbon atoms, mercapto, alkylthio with 1 to 4 carbon atoms, halogen(preferably fluorine, chlorine and bromine) or nitro);

cycloalkyl, preferably a carbocyclic radical with 3 to 7 ring carbonatoms (preferably 5 to 7 ring carbon atoms), which can be substituted,(e.g. the groups and atoms mentioned above in the case of open-chainhydrocarbon radicals R);

aryl is preferably a monocyclic or bicyclic aromatic radical with 6 to10 carbon atoms in the aryl part (e.g. phenyl, biphenyl, naphthyl, etc.,in particular phenyl, which can be substituted), optionally substitutedaryl or aralkyl radicals, preferably 1 to 3 identical or differentsubstituents (e.g. alkyl with 1 to 10 carbon atoms, optionallysubstituted, (e.g. chlorine, nitro or cyano); optionally substitutedalkenyl radicals with 1 to 10 carbon atoms; hydroxyl or alkoxy withpreferably 1 to 4 carbon atoms; amino or monoalkylamino and dialkylaminowith preferably 1 to 4 carbon atoms per alkyl radical: mercapto oralkylthio with preferably 1 to 4 carbon atoms; and carboxyl orcarbalkoxy with preferably 1 to 4 carbon atoms; the sulphonic acidgroup, alkylsulphonyl with preferably 1 to 4 carbon atoms andarylsulphonyl, preferably phenylsulphonyl; aminosulphonyl oralkylaminosulphonyl and dialkylaminosulphonyl with 1 to 4 carbon atomsper alkyl group, preferably methylaminosulphonyl anddimethylaminosulphonyi; nitro, cyano or the aldehyde group;alkylcarbonylamino with preferably 1 to 4 carbon atoms; andalkylcarbonyl with 1 to 4 carbon atoms, benzoyl, benzylcarbonyl andphenethylcarbonyl, the last-mentioned alkyl; phenyl, benzyl andphenethyl radicals may be optionally substituted (e.g. chlorine, nitroor hydroxyl, as well as radicals derived from sugars);

aralkyl preferably has 6 to 10, especially 6, carbon atoms in the arylpart said aryl part being preferably monocyclic or bicyclic carbocyclicaryl, such as phenyl, biphenyl or naphthyl, and preferably 1 to 4,especially 1 or 2, carbon atoms in the alkyl part, as for example inbenzyl or phenylethyl. Possible substituents for the aryl part of thearalkyl radical are preferably those substituents mentioned for the arylradicals R above;

Heterocyclyl preferably has a hetero-paraffinic, heteroaromatic orhetero-olefinic 5-membered or 6-membered ring, with preferably 1 to 3identical or different hetero-atoms (e.g. oxygen, sulphur or nitrogen),optionally substituted (e.g. hydroxyl, amino, C₁-C₄-alkyl groups,benzene nuclei or further, preferably 6-membered, heterocyclic rings ofthe type mentioned can be fused to them, wherein the bonding of theheterocyclic radical R is effected via a carbon atom of the heterocyclicsystem or of the fused benzene nucleus (preferred heterocyclic radicalsare derived, e.g., from furan, pyran, pyrrolidine, piperidine, pyrazole,imidazole, pyrimidine, pyridazine, pyrazine, triazine, pyrrole,pyridine, benzimidazole, quinoline, isoquinoline or purine, includingthose heterocycles which are bonded via a —CH₂— bridge outside the ring,for example the furfuryl radical));

wherein R₁ of NRR¹, is alkyl, cycloalkyl, aralkyl, or aryl in which R₁preferably represents a straight-chain or branched alkyl radical with1-6 carbon atoms or a cycloalkyl, aralkyl or aryl radical as definedabove for R (e.g. cyclopentyl, cyclohexyl, benzyl or phenyl radical), itbeing possible for the radicals mentioned to be preferably substitutedby alkoxy with 1 to 4 carbon atoms, amino, C₁-C₄ monoalkylamino andC₁-C₄-dialkylamino, nitro, cyano, hydroxyl, mercapto, C₁-C₄-thioalkyl orthe carboxyl or sulphonic acid group, in the case where R₁ denotesphenyl, also by C₁-C₄-alkyl;

wherein R and R₁ and the nitrogen atom to which they are bonded, mayoptionally form a heterocyclic ring, optionally saturated orunsaturated, the ring optionally containing 1 to 3 further(preferably 1) oxygen atoms, sulphur atoms or nitrogen atoms and, ashetero groups, a SO₂ group or a N-alkyl group, the alkyl (e.g. methyl,ethyl, n- and i-propyl and n-, l- and t-butyl) in the N-alkyl grouppreferably containing 1-4, in particular 1 or 2, carbon atoms;

wherein the heterocyclic ring contains 5-7, preferably 5 or 6, ringmembers. The 6-membered heterocyclic ring preferably contains thehetero-atom or the hetero-group in the para-position relative to thenitrogen atom (e.g. pyrrolidine, piperidine, hexamethleneimine,morpholine and N-methylpiperazine).

J. Antibiotics 36 p1157-1165 (1983) discloses the fermentation andisolation of a family of amylase inhibitors, trestatin-A, B and C. J.Antibiotics 36 p1166-1175 (1983) discloses the structures oftrestatin-A, B and C

J. Antibiotics 37 p182-186 (1984) describes the isolation,characterisation and structure elucidation of higher homologues of thetrestatins. The structures disclosed are:

The amylase inhibitor, V-1532, is prepared and characterised asdescribed in J. Mol. Biol. 260, 409-421, (1996).

Chem. Pharm. Bull 47(2), 187-193 (1999) describes the synthesis of thefollowing N-containing maltooligosaccharides with α-amylase activity.

R n Chem. Pharm Bull Ref. Number A 0 Compound 6 A 1 Compound 7 A 2Compound 8 A 3 Compound 9 A 4 Compound 10 B 0 Compound 11 B 1 Compound12 B 2 Compound 13 B 3 Compound 14 B 4 Compound 15 C 0 Compound 16 C 1Compound 17 C 2 Compound 18 C 3 Compound 19 C 4 Compound 20 D 1 Compound22 D 2 Compound 23 D 3 Compound 24

Agric. Biol. Chem, 41(11) 2221-2228 (1977) describes the fermentation,recovery and isolation of the microbial natural product amylaseinhibitor, SA-1. Although the structure of SA-1 is unknown, the compoundhas been shown to be homogeneous by tlc and is characterised byanalytical data.

Kor. J. Mycol. Vol 13, No. 4, 203-212, (1985) describes the fermentationand purification of a microbial natural product □-amylase inhibitor fromculture filtrates of Streptomyces strain DMC-72. The compound ischaracterised by analytical data.

EP-194794 (WO-8605094 PCT equivalent) reports the structures of a numberof N-substituted valiolamine derivatives, referring to EP-56194 fortheir synthesis. The compounds have the structure:

in which A is an acyclic hydrocarbon group of 1 to 10 carbon atoms whichmay have one or more members selected from the group consisting ofhydroxy, phenoxy, thienyl, furyl, pyridyl, cyclohexyl, and a substitutedor unsubstituted phenyl; a five- to six-membered cyclic hydrocarbongroup which may have one or more members selected from the groupconsisting of hydroxy, hydroxymethyl, methyl and amino, or a saccharideresidue.ES-8800955 describes valiolamine and validamine analogues with thestructures:

in which A is a hydrocarbon group of 1 to 10 carbon atoms, optionallysubstituted with hydroxy, phenoxy, thienyl, furyl, pyridyl, cyclohexyl;or a phenyl group optionally substituted; or a cyclic hydrocarbon of 3-7carbon atoms, optionally substituted with hydroxyl, and B is hydrogen orhydroxyl.

EP-301 400 (US equivalent—U.S. Pat. No. 4,885,361) describes thesulphation of the trestatins to give sulphated oligosaccharides withstructures:

wherein n is a whole number from 1-3; R is hydrogen or a residue —SO₃Mand M is a cation; and in which the degree of sulphation is at least 1.

EP-173950 describes the fermentation, recovery and isolation of thepseudooligosaccharide α-glycosidase inhibitor from Streptomyces sp.FH-1717 (DSM-3006). This compound has the structure shown:

EP-49981 discloses the synthesis of some N-substituted valienaminederivatives:

in which A is a chain hydrocarbon group having 1 to 10 carbon atomsoptionally substituted by hydroxyl, phenoxy, thienyl, furyl, pyridyl,cyclohexyl or phenyl optionally substituted by hydroxyl, lower alkoxy,lower alkyl, halogen or carboxyl; or a cyclic hydrocarbon group having 3to 7 carbon atoms optionally substituted by hydroxyl.

Angewandte Chemie Int. Ed. 20, 744-761 (1981) reviews the chemistry ofmicrobial derived α-glucosidase inhibitors. The oligosaccharides aredescribed elsewhere in this specification. The properties of the lowmolecular weight inhibitors, nojirimycin and 1-deoxynojirimycin, arereported.

The fermentation, recovery, resin and HPLC purification, and nmrassignment of an oligosaccharide amylase inhibitor from Streptomycesconglobatus, ATCC-31005 is described in Example 7 of the Experimentalsection of this specification.

The fermentation, recovery, resin and HPLC purification, and nmrassignment of a novel oligosaccharide amylase inhibitor fromStreptomyces conglobatus, ATCC-31005 is described in Example 8 of theExperimental section of this specification.

Tetrahedron Letters, 37, 14, 2479-2482 (1996) describes the synthesis ofβ-acarbose from 1-epivalienamine.

Both isoacarbose and acarviosine-glucose can be produced by the enzymictransformation of acarbose, as reported in Archives Biochem. Biophys,371, 2, 277-283 (1999).

The synthesis of adiposin-2 is reported in JCS Chem. Comm., 9, 605-606(1988)

Derivatives of the above compounds transformed as follows: 1. All abovecompounds with a valieneamine moiety can be transformed into thesaturated analogue, produced by a reduction process described forexample in EP-67356

2. The chain extended, viz.: see EP-240175-A

see CH-648-326-A

see EP-89812-A Other derivatives incorporating the the valineamine coremoiety, and of all compounds mentioned above, specifically acarbose,higher homologues thereof, trestatins, and V-1532, and the valeineaminecompounds given by their registry numbers below* can be made by methodsdescribed herein.

certain compounds with the moiety shown above appear in ChemicalAbstracts* with the Registry Numbers (RN) shown below.

Stick, Robert V.; Tilbrook, D. Matthew G.; Williams, Spencer J.Australian Journal of Chemistry (1999), 52(9), 895-904. p896.

Stick, Robert V.; Tilbrook, D. Matthew G.; Williams, Spencer J.Australian Journal of Chemistry (1999), 52(9), 895-904 p896.

Crueger, Anneliese; Doerschug, Michael; Heiker, Fred-Robert; Von Hugo,Hasso; Rauenbusch, Erich. DE-19821038-A1 p2.

Mahmud, Taifo; Tornus, Ingo; Egelkrout, Erin; Wolf, Eckardt; Uy,Charmaine; Floss, Heinz G.; Lee, Sungsook. Journal of the AmericanChemical Society (1999), 121(30), 6973-6983.

Park, Kwan Hwa; Kim, Myo Jeong; Lee, Hee Seob; Han, Nam Soo; Kim, Doman;Robyt, John F. Carbohydrate Research (1998), 313(3-4), 235-246.

Payre, Nathalie; Cottaz, Sylvain; Boisset, Claire; Borsali, Redouane;Svensson, Birte; Henrissat, Bernard; Driguez, Hugues. Angewandte Chemie,International Edition (1999), 38(7), 974-977. p975.

Lee, Sungsook; Tornus, Ingo; Dong, Haijun; Groger, Stefan. Journal ofLabelled Compounds & Radiopharmaceuticals (1999), 42(4), 361-372 p363.

Lee, Sungsook; Tornus, Ingo; Dong, Haijun; Groger, Stefan. Journal ofLabelled Compounds & Radiopharmaceuticals (1999), 42(4), 361-372 p363.

Shing, Tony K. M.; Li, Tin Y.; Kok, Stanton H.-L. Department ofChemistry, The Chinese University of Hong Kong, Shatin, Peop. Rep.China. Journal of Organic Chemistry (1999), 64(6), 1941-1946. compound 2p1942.

Sigurskjold, Bent W.; Christensen, Trine; Payre, Nathalie; Cottaz,Sylvain; Driguez, Hugues; Svensson, Birte. Biochemistry (1998), 37(29),10446-10452. structure referenced on page 10448.

Ogawa, Seiichiro; Ashiura, Makoto; Uchida, Chikara. CarbohydrateResearch (1998), 307(1,2), 83-95. compound 37 p88.

Ogawa, Seiichiro; Ashiura, Makoto; Uchida, Chikara. CarbohydrateResearch (1998), 307(1,2), 83-95. compound 5 p88.

McAuliffe, Joseph C.; Stick, Robert V.; Matthew, D.; Tilbrook, G.;Watts, Andrew G. Department of Chemistry, The University of WesternAustralia, Nedlands, Australia. Australian Journal of Chemistry (1998),51(2), 91-95. compound 3 p91.

Crueger, Anneliese; Dellweg, Hans-Georg; Lenz, Juergen Georg; Schroeder,Werner; Pape, Hermann; Goeke, Klaus; Schaper, Beate; Hemker, Michael;Piepersberg, Wolfgang; Distler, Juergen; Stratmann, Ansgar. EP-796915-A2p13.

Ogawa, Seiichiro; Mito, Tamami; Taiji, Eiichi; Jimbo, Masayuki;Yamagishi, Kiwamu; Inokuchi, Jin-Ichi. Bioorganic & Medicinal ChemistryLetters (1997), 7(14), 1915-1920. compound 16b p1917.

Ogawa, Seiichiro; Mito, Tamami; Taiji, Eiichi; Jimbo, Masayuki;Yamagishi, Kiwamu; Inokuchi, Jin-Ichi. Bioorganic & Medicinal ChemistryLetters (1997), 7(14). 1915-1920. compound 15b p1917.

Ogawa, Seiichiro; Mito, Tamami; Taiji, Eiichi; Jimbo, Masayuki;Yamagishi, Kiwamu; Inokuchi, Jin-Ichi. Bioorganic & Medicinal ChemistryLetters (1997), 7(14), 1915-1920. compound 6a p1916.

Ogawa, Seiichiro; Mito, Tamami; Taiji, Eiichi; Jimbo, Masayuki;Yamagishi, Kiwamu; Inokuchi, Jin-Ichi. Bioorganic & Medicinal ChemistryLetters (1997), 7(14), compound 4d p1916.

Ogawa, Seiichiro; Mito, Tamami; Taiji, Eiichi; Jimbo, Masayuki;Yamagishi, Kiwamu; Inokuchi, Jin-Ichi. Bioorganic & Medicinal ChemistryLetters (1997), 7(14), compound 4c p1916.

Ogawa, Seiichiro; Mito, Tamami; Taiji, Eiichi; Jimbo, Masayuki;Yamagishi, Kiwamu; Inokuchi, Jin-Ichi. Bioorganic & Medicinal ChemistryLetters (1997), 7(14), compound 4b p1916.

Ogawa, Seiichiro; Mito, Tamami; Taiji, Eiichi; Jimbo, Masayuki;Yamagishi, Kiwamu; Inokuchi, Jin-Ichi. Bioorganic & Medicinal ChemistryLetters (1997), 7(14), compound 4a p1916.

McAuliffe, Joseph C.; Stick, Robert V. Australian Journal of Chemistry(1997), 50(3), 219-224 compound 27 p220.

McAuliffe, Joseph C.; Stick, Robert V. Australian Journal of Chemistry(1997), 50(3), 225-228 compound 2 p226.

McAuliffe, Joseph C.; Stick, Robert V. Australian Journal of Chemistry(1997), 50(3), 203-207.

McAuliffe, Joseph C.; Stick, Robert V. Australian Journal of Chemistry(1997), 50(3), 203-207.

O. Srivastava and R. Sweda; U.S. Pat. No. 5,929,037 example 45 column30.

O. Srivastava and R. Sweda; U.S. Pat. No. 5,929,037 example 44 column29.

Banks et. al. EP-1157696-A2 p24.

Ogawa, Seiichiro; Ashiura, Makoto; Uchida, Chikara; Watanabe, Shinsuke;Yamazaki, Chihiro; Yamagishi, Kimamu; Inokuchi. Jin-ichi. Bioorganic &Medicinal Chemistry Letters (1996), 6(8), 929-932 compound 3a p929.

Ogawa, Seiichiro; Ashiura, Makoto; Uchida, Chikara; Watanabe, Shinsuke;Yamazaki, Chihiro; Yamagishi, Kimamu; Inokuchi, Jin-ichi. Bioorganic &Medicinal Chemistry Letters (1996), 6(8), 929-932 compound 3f p929.

Ogawa, Seiichiro; Ashiura, Makoto; Uchida, Chikara; Watanabe, Shinsuke;Yamazaki, Chihiro; Yamagishi, Kimamu; Inokuchi, Jin-ichi. Bioorganic &Medicinal Chemistry Letters (1996), 6(8), 929-932 compound 3e p929.

Ogawa, Seiichiro; Ashiura, Makoto; Uchida, Chikara; Watanabe, Shinsuke;Yamazaki, Chihiro; Yamagishi, Kimamu; Inokuchi, Jin-ichi. Bioorganic &Medicinal Chemistry Letters (1996), 6(8), 929-932 compound 3d p929.

Ogawa, Seiichiro; Ashiura, Makoto; Uchida, Chikara; Watanabe, Shinsuke;Yamazaki, Chihiro; Yamagishi, Kimamu; Inokuchi, Jin-ichi. Bioorganic &Medicinal Chemistry Letters (1996), 6(8), 929-932 compound 3c p929.

Ogawa, Seiichiro; Ashiura, Makoto; Uchida, Chikara; Watanabe, Shinsuke;Yamazaki, Chihiro; Yamagishi, Kimamu; Inokuchi, Jin-ichi. Bioorganic &Medicinal Chemistry Letters (1996), 6(8), 929-932 compound 3b p929.

McAuliffe, Joseph C.; Stick, Robert V.; Stone, Bruce A. TetrahedronLetters (1996), 37(14), 2479-82 compound □epimer of 2b p2479.

McAuliffe, Joseph C.; Stick, Robert V.; Stone, Bruce A. TetrahedronLetters (1996), 37(14), 2479-82 compound α-epimer of 2b p2479.

McAuliffe, Joseph C.; Stick, Robert V.; Stone, Bruce A. TetrahedronLetters (1996), 37(14), 2479-82 compound 16 p2480.

Cassset, Florence; Imberty, Anne; Haser, Richard; Payan, Francoise;Perez, Serge. European Journal of Biochemistry (1995), 232(1), 284-93.compound a p286.

Ogawa, Seiichiro; Sasaki, Shin-ichi; Tsunoda, Hidetoshi. CarbohydrateResearch (1995), 274 183-96 compound 4 p185.

Uchida, Chikara; Kitahashi, Hideo; Watanabe, Shinsuke; Ogawa, Seiichiro.Journal of the Chemical Society, Perkin Transactions 1: Organic andBio-Organic Chemistry (1995), (13), 1707-17 compound 10 p1708.

Tsunoda, Hidetoshi; Inokuchi, Jinichi; Yamagishi, Kiwamu; Ogawa,Seiichiro. Liebigs Annalen (1995), (2), 279-84 279.

Tsunoda, Hidetoshi; Inokuchi, Jinichi; Yamagishi, Kiwamu; Ogawa,Seiichiro. Liebigs Annalen (1995), (2), 279-84 p279.

Tsunoda, Hidetoshi; Inokuchi, Jinichi; Yamagishi, Kiwamu; Ogawa,Seiichiro. Liebigs Annalen (1995), (2), 279-84 p279.

Tsunoda, Hidetoshi; Inokuchi, Jinichi; Yamagishi, Kiwamu; Ogawa,Seiichiro. Liebigs Annalen (1995), (2), 279-84 p279.

Tsunoda, Hidetoshi; Inokuchi, Jinichi; Yamagishi, Kiwamu; Ogawa,Seiichiro. Liebigs Annalen (1995), (2), 279-84 p280.

Ogawa, Seiichiro; Tsunoda, Hidetoshi; Inokuchi, Jinichi. Journal of theChemical Society, Chemical Communications (1994), (11), 1317-18 compoundE-2 p1317.

Ogawa, Seiichiro; Tsunoda, Hidetoshi; Inokuchi, Jinichi. Journal of theChemical Society, Chemical Communications (1994), (11), 1317-18 compound11 p1317.

Ogawa, Seiichiro; Tsunoda, Hidetoshi; Inokuchi, Jinichi. Journal of theChemical Society, Chemical Communications (1994), (11), 1317-18.compound Z-2 p1317.

Ishiguro, Toshihiro; Oka, Masahide; Yamaguchi, Takamasa; Nogami, Ikuo.EP-599646-A2 p11 Table 4 row 6.

Ogawa, Seiichiro; Aso, Daisuke. Carbohydrate Research (1993), 250(1),177-84. compound 4 p178.

Ogawa, Seiichiro; Aso, Daisuke. Carbohydrate Research (1993), 250(1),177-84. compound 11 p178.

Ogawa, Seiichiro; Aso, Daisuke. Carbohydrate Research (1993), 250(1),177-84. compound 9 p178.

Ogawa, Seiichiro; Aso, Daisuke. Carbohydrate Research (1993), 250(1),177-84. compound 10 p178.

Ogawa, Seiichiro; Aso, Daisuke. Carbohydrate Research (1993), 250(1),177-84. compound 8 p178.

Ogawa, Seiichiro; Aso, Daisuke. Carbohydrate Research (1993), 250(1),177-84. compound 7 p178.

Ogawa, Seiichiro; Aso, Daisuke. Carbohydrate Research (1993). 250(1),177-84. compound 6 p178.

Ogawa, Seiichiro; Aso, Daisuke. Carbohydrate Research (1993), 250(1),177-84. compound 5 p178.

Cottaz, Sylvain; Brimacombe, John S.; Ferguson, Michael A. J.Carbohydrate Research (1993), 247 341-5.

Ogawa, Seiichiro; Sato, Koji; Miyamoto, Yasunobu. Journal of theChemical Society, Perkin Transactions 1: Organic and Bio-Organic Chem.(1972-1999) (1993), (6), 691-6.

Shibata, Yasushi; Kosuge, Yasuhiro; Mizukoshi, Toshimi; Ogawa,Seiichiro. Carbohydrate Research (1992), 228(2), 377-98. compound 9p378.

Shibata, Yasushi; Kosuge, Yasuhiro; Mizukoshi, Toshimi; Ogawa,Seiichiro. Carbohydrate Research (1992), 228(2), 377-98. compound 8p378.

Shibata, Yasushi; Kosuge, Yasuhiro; Mizukoshi, Toshimi; Ogawa,Seiichiro. Carbohydrate Research (1992), 228(2), 377-98. compound 7p378.

Shibata, Yasushi; Kosuge, Yasuhiro; Mizukoshi, Toshimi; Ogawa,Seiichiro. Carbohydrate Research (1992), 228(2), 377-98. compound 6p378.

Shibata, Yasushi; Kosuge, Yasuhiro; Mizukoshi, Toshimi; Ogawa,Seiichiro. Carbohydrate Research (1992), 228(2), 377-98. compound 5p378.

Shibata, Yasushi; Kosuge, Yasuhiro; Mizukoshi, Toshimi; Ogawa,Seiichiro. Carbohydrate Research (1992), 228(2), 377-98. compound 4p378.

Shibata, Yasushi; Kosuge, Yasuhiro; Mizukoshi, Toshimi; Ogawa,Seiichiro. Carbohydrate Research (1992), 228(2), 377-98. compound 3p378.

Svensson, Birte; Sierks, Michael R. Dep. Chem., Carlsberg Lab., Valby,Den. Carbohydrate Research (1992), 227 p29-44.

Shibata Y; Kosuge Y; Mizukoshi T; Ogawa S Carbohydrate Research (1992Apr. 27), 228(2). 377-98 compound 1 p378.

Ogawa, Seiichiro; Nakamura, Yoshikazu. Fac. Sci. Technol., Keio Univ.,Yokohama, Japan. Carbohydrate Research (1992), 226(1), 79-89 compound 5ap79.

Ogawa, Seiichiro; Nakamura, Yoshikazu. Fac. Sci. Technol., Keio Univ.,Yokohama, Japan. Carbohydrate Research (1992), 226(1), 79-89 compound 4ap79.

Ogawa, Seiichiro; Nakamura, Yoshikazu. Fac. Sci. Technol., Keio Univ.,Yokohama, Japan. Carbohydrate Research (1992), 226(1), 79-89 compound 3ap79.

Ogawa, Seiichiro; Uchida, Chikara; Shibata, Yasushi. CarbohydrateResearch (1992), 223 279-86.

Asano, Naoki; Kameda, Yukihiko; Matsui, Katsuhiko. Journal ofAntibiotics (1991), 44(12), 1406-16 p1407.

Asano, Naoki; Kameda, Yukihiko; Matsui, Katsuhiko. Journal ofAntibiotics (1991), 44(12). 1406-16 p1407.

Asano, Naoki; Kameda, Yukihiko; Matsui, Katsuhiko. Journal ofAntibiotics (1991), 44(12), 1406-16 p1407.

Asano, Naoki; Kameda, Yukihiko; Matsui, Katsuhiko. Journal ofAntibiotics (1991), 44(12), 1406-16 p1407.

Asano, Naoki; Kameda, Yukihiko; Matsui, Katsuhiko. Journal ofAntibiotics (1991), 44(12), 1406-16 p1409.

Furumoto, Tadashi; Yoshioka, Tadashi; Kamata, Kanae; Kameda, Yukihiko;Matsui, Katsuhiko. Journal of Antibiotics (1991), 44(3), 371-3.

Furumoto, Tadashi; Kameda, Yukihiko; Matsui, Katsuhiko. Chemical &Pharmaceutical Bulletin (1992), 40(7), 1871-5 p1872.

Ogawa, Seiichiro; Shibata, Yasushi; Kosuge, Yasuhiro; Yasuda, Kuninobu;Mizukoshi, Toshimi; Uchida, Chikara. Journal of the Chemical Society,Chemical Communications (1990), (20), 1387-8 p1388.

Ogawa, Seiichiro; Shibata, Yasushi; Kosuge, Yasuhiro; Yasuda, Kuninobu;Mizukoshi, Toshimi; Uchida, Chikara. Journal of the Chemical Society,Chemical Communications (1990), (20), 1387-8 p1388.

Ogawa, Seiichiro; Shibata, Yasushi; Kosuge, Yasuhiro; Yasuda, Kuninobu;Mizukoshi, Toshimi; Uchida, Chikara. Journal of the Chemical Society,Chemical Communications (1990), (20), 1387-8 p1388.

Ogawa, Seiichiro; Shibata, Yasushi; Kosuge, Yasuhiro; Yasuda, Kuninobu;Mizukoshi, Toshimi; Uchida, Chikara. Journal of the Chemical Society,Chemical Communications (1990), (20), 1387-8 p1388.

Asano, Naoki; Kameda, Yukihiko; Matsui, Katsuhiko; Horii, Satoshi;Fukase, Hiroshi. Journal of Antibiotics (1990), 43(8), 1039-41 p1040.

Wessel, Hans Peter; Hosang, Markus; Tschopp, Thomas B.; Weimann, BerndJuergen. Carbohydrate Research (1990), 204 131-9.

Boberg, M.; Kurz, J.; Ploschke, H. J.; Schmitt, P.; Scholl, H.;Shueller, M.; Wuensche, C. Arzneimittel-Forschung (1990), 40(5), 555-63compound 4 p559.

Boberg, M.; Kurz, J.; Ploschke, H. J.; Schmitt, P.; Scholl, H.;Shueller, M.; Wuensche, C. Arzneimittel-Forschung (1990), 40(5), 555-63p559.

Vertesy et. al. U.S. Pat. No. 5,091,524.

Nicotra, Francesco; Panza, Luigi; Ronchetti, Fiamma; Russo, Giovanni.Gazzetta Chimica Italiana (1989), 119(11), 577-9 compound 2 p577.

Maul, W.; Mueller, L.; Pfitzner, J.; Rauenbusch, E.; Schutt, H.Arzneimittel-Forschung (1989), 39(10), 1251-3 p1251.

Takahashi, Yoshinori; Sakaguchi, Fumiaki; Morimoto, Keiko; Hashimoto,Komei; Funaba, Tsukasa; Hayauchi, Yutaka. Iyakuhin Kenkyu (1989), 20(4),769-83.

Takahashi, Yoshinori; Sakaguchi, Fumiaki; Morimoto, Keiko; Hashimoto,Komei; Funaba, Tsukasa; Hayauchi, Yutaka. Iyakuhin Kenkyu (1989), 20(4),769-83.

Jin, Wen Zao; Rinehart, Kenneth L., Jr.; Toyokuni, Tatsushi. Journal ofAntibiotics (1987), 40(3), 329-39 compound 4 p336.

Jin, Wen Zao; Rinehart, Kenneth L.; Jr.; Toyokuni, Tatsushi. Journal ofAntibiotics (1987), 40(3), 329-39 compound 6 p337.

Ogawa, Seiichiro; Sugizaki, Hiroyasu. Chemistry Letters (1986), (11),1977-80 compound 5b p1978.

Ogawa, Seiichiro; Sugizaki, Hiroyasu. Chemistry Letters (1986), (11),1977-80 p1978.

Pfeffer, M.; Siebert, G. Zeitschrift fuer Ernaehrungswissenschaft(1986), 25(3), 189-95.

Pfeffer, M.; Siebert, G. Zeitschrift fuer Ernaehrungswissenschaft(1986), 25(3), 189-95.

Pfeffer, M.; Siebert, G. Zeitschrift fuer Ernaehrungswissenschaft(1986), 25(3), 189-95.

Pfeffer, M.; Siebert, G. Zeitschrift fuer Ernaehrungswissenschaft(1986), 25(3), 189-95.

Pfeffer, M.; Siebert, G. Zeitschrift fuer Ernaehrungswissenschaft(1986), 25(3), 189-95.

Fukase, Hiroshi; Horii, Satoshi. Journal of Organic Chemistry (1992),57(13), 3651-8.

Fukase, Hiroshi; Horii, Satoshi. Journal of Organic Chemistry (1992),57(13), 3651-8.

Kameda, Yukihiko; Asano, Naoki; Yamaguchi, Takuji; Matsui, Katsuhiko;Horii, Satoshi; Fukase, Hiroshi. Journal of Antibiotics (1986), 39(10),1491-4 p1491.

Kameda, Yukihiko; Asano, Naoki; Yamaguchi, Takuji; Matsui, Katsuhiko;Horii, Satoshi; Fukase, Hiroshi. Journal of Antibiotics (1986), 39(10),1491-4 p1491.

Vertesy, Laszlo; Bender, Rudolf; Fehlhaber, Hans Wolfram. EP-173950-A2p1.

Ogawa, Seiichiro; Miyamoto, Yasunobu; Nose, Taisuke. Journal of theChemical Society, Perkin Transactions 1: Organic and Bio-OrganicChemistry (1972-1999) (1988), (9), 2675-80 compound 1a p2675.

Ogawa, Seiichiro; Nose, Taisuke; Ogawa, Takao; Toyokuni, Tatsushi;Iwasawa, Yoshikazu; Suami, Tetsuo. Journal of the Chemical Society,Perkin Transactions 1: Organic and Bio-Organic Chemistry (1972-1999)(1985), (11), 2369-74.

Ogawa, Seiichiro; Nose, Taisuke; Ogawa, Takao; Toyokuni, Tatsushi;Iwasawa, Yoshikazu; Suami, Tetsuo. Journal of the Chemical Society,Perkin Transactions 1: Organic and Bio-Organic Chemistry (1972-1999)(1985), (11), 2369-74.

Ogawa, Seiichiro; Nose, Taisuke; Ogawa, Takao; Toyokuni, Tatsushi;Iwasawa, Yoshikazu; Suami, Tetsuo. Journal of the Chemical Society,Perkin Transactions 1: Organic and Bio-Organic Chemistry (1972-1999)(1985), (11), 2369-74.

Ogawa, Seiichiro; Iwasawa, Yoshikazu; Toyokuni, Tatsushi; Suami, Tetsuo.Carbohydrate Research (1985), 141(1), 29-40.

Ogawa, Seiichiro; Yasuda, Kuninobu; Takagaki, Tohei; Iwasawa, Yoshikazu;Suami, Tetsuo. Carbohydrate Research (1985), 141(2), 329-34 p330.

Ogawa, Seiichiro; Yasuda, Kuninobu; Takagaki, Tohei; Iwasawa, Yoshikazu;Suami, Tetsuo. Carbohydrate Research (1985), 141(2), 329-34.

Schmidt, Richard R.; Laesecke, Klaus. CH-648326-A.

Horii, Satoshi; Fukase, Hiroshi; Matsuo, Takao; Kameda, Yukihiko; Asano,Naoki; Matsui, Katsuhiko. Journal of Medicinal Chemistry (1986), 29(6),1038-46 p1040.

Horii, Satoshi; Fukase, Hiroshi; Matsuo, Takao; Kameda, Yukihiko; Asano,Naoki; Matsui, Katsuhiko. Journal of Medicinal Chemistry (1986), 29(6),1038-46 p1040.

Ogawa, Seiichiro; Iwasawa, Yoshikazu; Toyokuni, Tatsushi; Suami, Tetsuo.Carbohydrate Research (1985), 141(1), 29-40.

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-89812-A1.

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-89812-A1.

Yokose, Kazuteru; Ogawa, Mayumi; Ogawa, Kiyoshi. Journal of Antibiotics(1984), 37(2), 182-6.

Ogawa, Seiichiro; Inoue, Makoto; Iwasawa, Yoshikazu; Toyokuni, Tatsushi;Suami, Tetsuo. Chemistry Letters (1983), (7), 1085-8 p1085.

Ogawa, Seiichiro; Inoue, Makoto; Iwasawa, Yoshikazu; Toyokuni, Tatsushi;Suami, Tetsuo. Chemistry Letters (1983), (7), 1085-8 p1085.

Toyokuni, Tatsushi; Ogawa, Seiichiro; Suami, Tetsuo. Bulletin of theChemical Society of Japan (1983), 56(4), 1161-70 p1163.

Heiker, Fred Robert; Mueller, Lutz; Puls, Walter; Bischoff, Hilmar.EP-64635-A1.

Heiker, Fred Robert; Mueller, Lutz; Puls, Walter; Bischoff, Hilmar.EP-64635-A1.

Heiker, Fred Robert; Mueller, Lutz; Puls, Walter; Bischoff, Hilmar.EP-64635-A1.

Junge, Bodo; Heiker, Fred R.; Kurz, Juergen; Mueller, Lutz; Schmidt,Delf D.; Wuensche, Christian. Carbohydrate Research (1984), 128(2),235-68.

Heiker, Fred Robert; Mueller, Lutz; Puls, Walter; Bischoff, Hilmar.EP-64635-A1.

Heiker, Fred Robert; Mueller, Lutz; Puls, Walter; Bischoff, Hilmar.EP-64635-A1.

Heiker, Fred Robert; Mueller, Lutz; Puls, Walter; Bischoff, Hilmar.EP-64635-A1.

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-89812-A1.

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-89812-A1.

Ogawa, Seiichiro; Suetsugu, Masaru; Toyakuni, Tatsushi; Suami, Tetsuo.Nippon Kagaku Kaishi (1982), (10), 1721-6.

Ogawa, Seiichiro; Suetsugu, Masaru; Toyakuni, Tatsushi; Suami, Tetsuo.Nippon Kagaku Kaishi (1982), (10), 1721-6.

Kameda, Yukihiko; Asano, Naoki; Yoshikawa, Michiyo; Matsui, Katsuhiko;Horii, Satoshi; Fukase, Hiroshi. Journal of Antibiotics (1982), 35(11),1624-6.

Kameda, Yukihiko; Asano, Naoki; Yoshikawa, Michiyo; Matsui, Katsuhiko;Horii, Satoshi; Fukase, Hiroshi. Journal of Antibiotics (1982), 35(11),1624-6.

Kameda, Yukihiko; Asano, Naoki; Yoshikawa, Michiyo; Matsui, Katsuhiko;Horii, Satoshi; Fukase, Hiroshi. Journal of Antibiotics (1982), 35(11),1624-6.

Kameda, Yukihiko; Asano, Naoki; Yoshikawa, Michiyo; Matsui, Katsuhiko;Horii, Satoshi; Fukase, Hiroshi. Journal of Antibiotics (1982), 35(11),1624-6.

Kameda, Yukihiko; Asano, Naoki; Yoshikawa, Michiyo; Matsui, Katsuhiko;Horii, Satoshi; Fukase, Hiroshi. Journal of Antibiotics (1982), 35(11),1624-6.

Kameda, Yukihiko; Asano, Naoki; Yoshikawa, Michiyo; Matsui, Katsuhiko;Horii, Satoshi; Fukase, Hiroshi. Journal of Antibiotics (1982), 35(11),1624-6.

Kameda, Yukihiko; Asano, Naoki; Yoshikawa, Michiyo; Matsui, Katsuhiko;Horii, Satoshi; Fukase, Hiroshi. Journal of Antibiotics (1982), 35(11),1624-6.

Kangouri, Kunjo; Namiki, Shinjuro; Nagate, Takatoshi; Hara, Hiroshi;Sugita, Kazuhiko; Omura, Sadafumi. Journal of Antibiotics (1982), 35(9),1160-6.

Kangouri, Kunjo; Namiki, Shinjuro; Nagate, Takatoshi; Hara, Hiroshi;Sugita, Kazuhiko; Omura, Sadafumi. Journal of Antibiotics (1982), 35(9),1160-6.

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-56194-A1.

Fukuhara, Kenichi; Murai, Hidetsugu; Murao, Sawao. Agricultural andBiological Chemistry (1982) 46(7), 1941-5.

Yoon, Seung-Heon; Robyt, John F. Carbohydrate Research (2002), 337(6),509-516.

Nahoum, Virginie; Roux, Genevieve; Anton, Veronique; Rouge, Pierre;Puigserver, Antoine; Bischoff, Hilmar; Henrissat, Bernard; Payan,Francoise. Biochemical Journal (2000), 346(1), 201-208.

Kim, Myo-Jeong; Lee, Hee-Seob; Cho, Jin-Sook; Kim, Tae-Jip; Moon,Tae-Wha; Oh, Sang-Taek; Kim, Jung-Wan; Oh, Byung-Ha; Park, Kwan-Hwa.Biochemistry (2002), 41(29), 9099-9108.

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p10.

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p10.

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p10.

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p10.

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p8.

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p12.

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p7.

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p16.

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p11.

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p7.

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p24.

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p23.

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p23.

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p23.

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p23.

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p12

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p12

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p12

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p12

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p10

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p11

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p10

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p10

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p20

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p20

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p20

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p19

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p19

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p19

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p19

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p19

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p18

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p18

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p18

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p18

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p18

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p18

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p17

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p17

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p17

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p17

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p16

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p16

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p16

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p15

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p15

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p15

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p14

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p14

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p14

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p14

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p14

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p13

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p13

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p13

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p13

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p13

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p13

Horii, Satoshi; Kameda, Yukihiko; Fukase, Hiroshi. EP-49981-A1 p13

Meiji Seika Kaisha, Ltd., Japan JP-57024397-A2 p1

Ogawa, Seiichiro; Ogawa, Takao; Chida, Noritaka; Toyokuni, Tatsushi;Suami, Tetsuo. Chemistry Letters (1982), (5), 749-52 p751

Ogawa, Seiichiro; Ogawa, Takao; Chida, Noritaka; Toyokuni, Tatsushi;Suami, Tetsuo. Chemistry Letters (1982), (5), 749-52 p751

Ogawa, Seiichiro; Ogawa, Takao; Chida, Noritaka; Toyokuni, Tatsushi;Suami, Tetsuo. Chemistry Letters (1982), (5), 749-52 p751

Ogawa, Seiichiro; Toyokuni, Tatsushi; Iwasawa, Yoshikazu; Abe, Yasuo;Suami, Tetsuo. Chemistry Letters (1982), (3), 279-82 p279

Ogawa, Seiichiro; Toyokuni, Tatsushi; Iwasawa, Yoshikazu; Abe, Yasuo;Suami, Tetsuo. Chemistry Letters (1982), (3), 279-82 p280

Meiji Seika Kaisha, Ltd., Japan JP-57024397-A2 p1

Taisho Pharmaceutical Co., Ltd., Japan. JP-56125398-A2 p1

Taisho Pharmaceutical Co., Ltd., Japan. JP-56125398-A2 p1

Taisho Pharmaceutical Co., Ltd., Japan. JP-56125398-A2 p1

Taisho Pharmaceutical Co., Ltd., Japan. JP-56125398-A2 p1

Taisho Pharmaceutical Co., Ltd., Japan. JP-56125398-A2 p1

Taisho Pharmaceutical Co., Ltd., Japan. JP-56125398-A2 p1

Taisho Pharmaceutical Co., Ltd., Japan. JP-56125398-A2 p1

Taisho Pharmaceutical Co., Ltd., Japan. JP-56125398-A2 p1

Ogawa, Seiichiro; Toyakuni, Tatsushi; Suami, Tetsuo. Chemistry Letters(1981), (7), 947-50.

Ogawa, Seiichiro; Toyokuni, Tatsushi; Suami, Tetsuo. Chemistry Letters(1981), (7), 947-50.

Takeda Chemical Industries, Ltd., Japan JP-56012399

Asano, Naoki; Kameda, Yukihiko; Matsui, Katsuhiko. Journal ofAntibiotics (1991), 44(12), 1406-16 compound 3a p1407

Mueller, L.; Junge, B.; Frommer, W.; Schmidt, D.; Truscheit, E. Inst.Biochem., Bayer A.-G., Wuppertal, Fed. Rep. Ger. Editor(s): Brodbeck,Urs. Enzyme Inhibitors, Proc. Meet. (1980), 109-22. Publisher: VerlagChain p117

Junge, B.; Boeshagen, H.; Stoltefuss, J.; Mueller, L. Inst. Biochem.,Bayer A.-G., Wuppertal, Fed. Rep. Ger. Editor(s): Brodbeck, Urs. EnzymeInhibitors, Proc. Meet. (1980), 123-37. Publisher: Verlag Chem. Diagram15 p 135

Junge, B.; Boeshagen, H.; Stoltefuss, J.; Mueller, L. Inst. Biochem.,Bayer A.-G., Wuppertal, Fed. Rep. Ger. Editor(s): Brodbeck, Urs. EnzymeInhibitors, Proc. Meet. (1980), 123-37. Publisher: Verlag Chem Diagram16 p136

Junge, B.; Boeshagen, H.; Stoltefuss, J.; Mueller, L. Inst. Biochem.,Bayer A.-G., Wuppertal, Fed. Rep. Ger. Editor(s): Brodbeck, Urs. EnzymeInhibitors, Proc. Meet. (1980), 123-37. Publisher: Verlag Chem diagram16 p136

Junge, B.; Boeshagen, H.; Stoltefuss, J.; Mueller, L. Inst. Biochem.,Bayer A.-G., Wuppertal, Fed. Rep. Ger. Editor(s): Brodbeck, Urs. EnzymeInhibitors, Proc. Meet. (1980), 123-37. Publisher: Verlag Chem diagram15 p135

Junge, B.; Boeshagen, H.; Stoltefuss, J.; Mueller, L. Inst. Biochem.,Bayer A.-G., Wuppertal, Fed. Rep. Ger. Editor(s): Brodbeck, Urs. EnzymeInhibitors, Proc. Meet. (1980), 123-37. Publisher: Verlag Chem

Junge, B.; Boeshagen, H.; Stoltefuss, J.; Mueller, L. Inst. Biochem.,Bayer A.-G., Wuppertal, Fed. Rep. Ger. Editor(s): Brodbeck, Urs. EnzymeInhibitors, Proc. Meet. (1980), 123-37. Publisher: Verlag Chem diagram13 p134

Mueller, L.; Junge, B.; Frommer, W.; Schmidt, D.; Truscheit, E. Inst.Biochem., Bayer A.-G., Wuppertal, Fed. Rep. Ger. Editor(s): Brodbeck,Urs. Enzyme Inhibitors, Proc. Meet. (1980), 109-22. Publisher: VerlagChain

Ajinomoto Co., Inc., Japan. JP-55157595

Takeda Chemical Industries, Ltd., Japan JP-55133393

Hasegawa, Akira; Kobayashi, Toshiyuki; Hibino, Hideyuki; Kiso, Makoto.Dep. Agric. Chem., Gifu Univ., Gifu, Japan. Agricultural and BiologicalChemistry (1980), 44(1), 143-7 p144

Hasegawa, Akira; Kobayashi, Toshiyuki, Hibino, Hideyuki; Kiso, Makoto.Dep. Agric. Chem., Gifu Univ., Gifu, Japan. Agricultural and BiologicalChemistry (1980), 44(1), 143-7 p144

Hasegawa, Akira; Kobayashi, Toshiyuki; Hibino, Hideyuki; Kiso, Makoto.Dep. Agric. Chem., Gifu Univ., Gifu, Japan. Agricultural and BiologicalChemistry (1980), 44(1), 143-7 p144

Hasegawa, Akira; Kobayashi, Toshiyuki; Hibino, Hideyuki; Kiso, Makoto.Dep. Agric. Chem., Gifu Univ., Gifu, Japan. Agricultural and BiologicalChemistry (1980), 44(1). 143-7 p144

Kameda Yukihiko. Takeda Chemical Industries, Ltd., Japan JP-55000308

Y. Suhara et. al. U.S. Pat. No. 4,273,765

Y. Suhara et. al. U.S. Pat. No. 4,273,765

Y. Suhara et. al. U.S. Pat. No. 4,273,765

Y. Suhara et. al. U.S. Pat. No. 4,273,765

Otani, Masaru; Saito, Tetsu; Satoi, Shuzo; Mizoguchi, Junzo; Muto,Naoki. Toyo Jozo Co., Ltd., Japan DE-2855409

Otani, Masaru; Saito, Tetsu; Satoi, Shuzo; Mizoguchi, Junzo; Muto,Naoki. Toyo Jozo Co., Ltd., Japan DE-2855409

Otani, Masaru; Saito, Tetsu; Satoi, Shuzo; Mizoguchi, Junzo; Muto,Naoki. Toyo Jozo Co., Ltd., Japan DE-2855409

Otani, Masaru; Saito, Tetsu; Satoi, Shuzo; Mizoguchi, Junzo; Muto,Naoki. Toyo Jozo Co., Ltd., Japan DE-2855409

E. Rauenbusch et. al. U.S. Pat. No. 4,174,439

B. Junge et. al. DE-2658562 p65

Kameda, Yukihiko; Asano, Naoki; Hashimoto, Tadashi. Journal ofAntibiotics (1978), 31(9), 936-8. p 936

Kameda, Yukihiko; Asano, Naoki; Hashimoto, Tadashi. Journal ofAntibiotics (1978), 31(9), 936-8. p936

Kameda, Yukihiko; Asano, Naoki; Hashimoto, Tadashi. Journal ofAntibiotics (1978), 31(9), 936-8. p937

B. Junge et. al. DE-2658562 p57

B. Junge et. al. DE-2658562 p27

B. Junge et. al. DE-2658562

B. Junge et. al. DE-2658562 p69

B. Junge et. al. DE-2658562 p69

B. Junge et. al. DE-2658562 p62

B. Junge et. al. DE-2658562 p31

B. Junge et. al. DE-2658562

B. Junge et. al. DE-2658562 p73

Junge, B.; Boeshagen, H.; Stoltefuss, J.; Mueller, L. Inst. Biochem.,Bayer A.-G., Wuppertal, Fed. Rep. Ger. Editor(s): Brodbeck, Urs. EnzymeInhibitors, Proc. Meet. (1980), 123-37. Publisher: Verlag Chem. Diagram16 p 136

Junge, B.; Boeshagen, H.; Stoltefuss, J.; Mueller, L. Inst. Biochem.,Bayer A.-G., Wuppertal, Fed. Rep. Ger. Editor(s): Brodbeck, Urs. EnzymeInhibitors, Proc. Meet. (1980), 123-37. Publisher: Verlag Chem. Diagram16 p 136

B. Junge et. al. DE-2658562 p73

B. Junge et. al. DE-2658562 p73

B. Junge et. al. DE-2658562 p72

B. Junge et. al. DE-2658562

Junge, B.; Boeshagen, H.; Stoltefuss, J.; Mueller, L. Inst. Biochem.,Bayer A.-G., Wuppertal, Fed. Rep. Ger. Editor(s): Brodbeck, Urs. EnzymeInhibitors, Proc. Meet. (1980), 123-37. Publisher: Verlag Chem. Diagram15 p 135

B. Junge et. al. DE-2658562 p72

B. Junge et. al. DE-2658562 p71

B. Junge et. al. DE-2658562 p71

B. Junge et. al. DE-2658562 p70

Junge, B.; Boeshagen, H.; Stoltefuss, J.; Mueller, L. Inst. Biochem.,Bayer A.-G., Wuppertal, Fed. Rep. Ger. Editor(s): Brodbeck, Urs. EnzymeInhibitors, Proc. Meet. (1980), 123-37. Publisher: Verlag Chem. P127

B. Junge et. al. DE-2658562 p63

B. Junge et. al. DE-2658562 p63

B. Junge et. al. DE-2658562 p63

B. Junge et. al. DE-2658562 p61

B. Junge et. al. DE-2658562 p61

B. Junge et. al. DE-2658562 p59

B. Junge et. al. DE-2658562 p59

B. Junge et. al. DE-2658562 p70

Junge, B.; Boeshagen, H.; Stoltefuss, J.; Mueller, L. Inst. Biochem.,Bayer A.-G., Wuppertal, Fed. Rep. Ger. Editor(s): Brodbeck, Urs. EnzymeInhibitors, Proc. Meet. (1980), 123-37. Publisher: Verlag Chem. Diagram14 p 135

B. Junge et. al. DE-2658562

B. Junge et. al. DE-2658562 p53

B. Junge et. al. DE-2658562 p57

B. Junge et. al. DE-2658562 p57

B. Junge et. al. DE-2658562 p56

B. Junge et. al. DE-2658562 p56

B. Junge et. al. DE-2658562 p56

B. Junge et. al. DE-2658562 p55

B. Junge et. al. DE-2658562 p55

B. Junge et. al. DE-2658562 p54

B. Junge et. al. DE-2658562 p54

B. Junge et. al. DE-2658562 p53

Frommer, Werner; Junge, Bodo; Keup, Uwe; Mueller, Lutz; Puls, Walter;Schmidt, Delf. U.S. Pat. No. 4,062,950 example 14

Frommer, Werner; Junge, Bodo; Keup, Uwe; Mueller, Lutz; Puls, Walter;Schmidt, U.S. Pat. No. 4,062,950 example 15

Asano N; Takeuchi M; Kameda Y; Matsui K; Kono Y JOURNAL OF ANTIBIOTICS(1990 June), 43(6), 722-6 p723

Asano N; Takeuchi M; Kameda Y; Matsui K; Kono Y JOURNAL OF ANTIBIOTICS(1990 June), 43(6), 722-6 p723

Kameda, Yukihiko; Horii, Satoshi. Journal of the Chemical Society,Chemical Communications (1972), (12), 746-7 p746

Chen, Xiaolong; Fan, Yongxian; Zheng, Yuguo; Shen, Yinchu. ChemicalReviews (Washington, D.C., United States) (2003), 103(5), 1955-1977

Horii, Satoshi; Kameda, Yukihiko. Journal of the Chemical Society,Chemical Communications (1972), (12), 747-8.

Horii, Satoshi; Kameda, Yukihiko; Kawahara, Kunio. Journal ofAntibiotics (1972), 25(1), 48-53 p51

Horii, Satoshi; Kameda, Yukihiko; Kawahara, Kunjo. Journal ofAntibiotics (1972), 25(1), 48-53 p51

Kim, Jung Woo; Lee, Kwang Moo; Chun, Hyoung Sik; Kim, Jong Gwan; Chang,Hung Bae; Kim, Sun Ho; Min, Kyeong Bok; Moon WO-9620945-A1 p10

Kim, Jung Woo; Lee, Kwang Moo; Chun, Hyoung Sik; Kim, Jong Gwan; Chang,Hung Bae; Kim, Sun Ho; Min, Kyeong Bok; Moon WO-9620945-A1 p10

Kim, Jung Woo; Lee, Kwang Moo; Chun, Hyoung Sik; Kim, Jong Gwan; Chang,Hung Bae; Kim, Sun Ho; Min, Kyeong Bok; Moon WO-9620945-A1 p10

Kim, Jung Woo; Lee, Kwang Moo; Chun, Hyoung Sik; Kim, Jong Gwan; Chang,Hung Bae; Kim, Sun Ho; Min, Kyeong Bok; Moon WO-9620945-A1 p10

Kim, Jung Woo; Lee, Kwang Moo; Chun, Hyoung Sik; Kim, Jong Gwan; Chang,Hung Bae; Kim, Sun Ho; Min, Kyeong Bok; Moon WO-9620945-A1 p10

Kim, Jung Woo; Lee, Kwang Moo; Chun, Hyoung Sik; Kim, Jong Gwan; Chang,Hung Bae; Kim, Sun Ho; Min, Kyeong Bok; Moon WO-9620945-A1 p10

Hata, Yoji; Kawato, Shoji; Abe, Yasuhisa; Ono, Kazuhisa. JP-02092267-A2

Vertesy, Laszlo; Betz, Joachim; Fehlhaber, Hans Wolfram; Geisen, Karl.EP-257418-A2

Vertesy, Laszlo; Betz, Joachim; Fehlhaber, Hans Wolfram; Geisen, Karl.EP-257418-A2

Ogawa, Seiichiro; Ikeda, Nobuo; Takeda, Haruki; Nakagawa, Yoshio.Carbohydrate Research (1988), 175(2), 294-301 p295

Shibata, Yasushi; Kosuge, Yasuhiro; Ogawa, Seiichiro. CarbohydrateResearch (1990), 199(1), 37-54p38

Shibata, Yasushi; Kosuge, Yasuhiro; Ogawa, Seiichiro. CarbohydrateResearch (1990), 199(1), 37-54p38

Shibata, Yasushi; Kosuge, Yasuhiro; Ogawa, Seiichiro. CarbohydrateResearch (1990), 199(1), 37-54p38

Shibata, Yasushi; Kosuge, Yasuhiro; Ogawa, Seiichiro. CarbohydrateResearch (1990), 199(1), 37-54 p38

Shibata, Yasushi; Kosuge, Yasuhiro; Ogawa, Seiichiro. CarbohydrateResearch (1990), 199(1), 37-54 p38

Shibata, Yasushi; Kosuge, Yasuhiro; Ogawa, Seiichiro. CarbohydrateResearch (1990), 199(1), 37-54 p38

Fujinomori, Kenichi; Ishikawa, Akira; Nishimoto, Mayumi. JP-10101507-A2

Ogawa, Seiichiro; Toyokuni, Tatsushi; Iwasawa, Yoshikazu; Abe, Yasuo;Suami, Tetsuo Chemistry Letters (1982), (3), 279-82

Ogawa, Seiichiro; Toyokuni, Tatsushi; Iwasawa, Yoshikazu; Abe, Yasuo;Suami, Tetsuo. Chemistry Letters (1982), (3), 279-82

Itoh J; Omoto S; Shomura T; Ogino H; Iwamatsu K; Inouye S; Hidaka H. J.Antibiotics (1981 November), 34(11), p1429

Itoh J; Omoto S; Shomura T; Ogino H; Iwamatsu K; Inouye S; Hidaka H J.Antibiotics (1981 November), 34(11), p1429

Itoh J; Omoto S; Shomura T; Ogino H; Iwamatsu K; Inouye S; Hidaka H. J.Antibiotics (1981 November), 34(11), p1429

Horii, Satoshi; Kameda, Yukihiko; Iwasa, Takashi; Yamamoto, Hitoichi.GB-1392505

Kameda, Yukihiko; Asano, Naoki; Yoshikawa, Michiyo; Takeuchi, Masayoshi;Yamaguchi, Takuji; Matsui, Katsuhiko; Horii, Satoshi; Fukase, Hiroshi.Journal of Antibiotics (1984), 37(11), 1301-7p1301.

By “acarbose and the higher homologues thereof” is meant theamylostatins of the formula given below, and mentioned generically andspecifically in British Patent No. GB 1,482,543; U.S. Pat. No.4,175,123; and in Agric. Biol. Chem., 46(7), 1941-1945, 1982, al ofwhich are hereby incorporated by reference in their entirety.

Compounds disclosed generically and specifically in GB1,482,543; U.S.Pat. No. 4,175,123; and in Agric. Biol. Chem.,46 (7), 1941-1945, 1982defined as “acarbose and higher homologues”* compounds where M = 0 and N= 1, 2 or 3 are disclosed in GB 1,482,543; compounds where M = 0 to 8,and the sum of M + N is 0 to 7; X in both cases is OR, SH, SR, NH₂, NHR,or NRR¹, where R is alkyl, alkenyl, cycloalkyl, aralkyl, aryl orheterocyclyl and is defined in the quoted patents.

In addition to the amylase and/or α-glucosidase inhibitor compoundsmentioned above, certain derivatives of said compounds can be madefollowing the types of chemical transformation disclosed in the tablesand references below, depending on the suitability of the substrate, andwhich transformations are expected to result in further amylase- and/orglucosidase-inhibiting substances.

Preferably the substrate for such transformation is selected from theamylostatin compounds (i.e “acarbose and higher homologues” mentionedabove), and trestatin compounds, V1532, the fraction 21 compound fromExample 7, the Example 8 compound, and the compounds shown below (orsuitably protected derivatives thereof):

PROCESS LITERATURE REF. (e.g.) EXAMPLES OF REACTING GROUPS. Synthetic orCH-648-326-A Any monosaccharide or oligosaccahride biotransformationJ.Chem. Soc. such as glucose, ribose, xylose, mannose, attachment of aPerkin Trans. 1 galactose, sucrose, etc. Any saccharide unit or (1982)1, pp 15-18 monosaccharide of 2-6 sugar monomer oligosaccaharide viaunits linked via any O or S for thio-sugars or a N, S or O atom N foraza-sugars Carbohydrate Any cyclitol such as those described in Research(1978) Cyclitols and their derivatives, 67, 2, pp 305-328 Hudlicky T,(1993), VCH publishers, Inc., New York Carbohydrate AlsoGlucose-O-benzene-OH (attached via Research (1997) any oxygen) andGlucose-O-benzene-O-glucose, 305, 3-4, pp 561-568 i.e which can beproduced by methods exemplified in and see also later Agric. Biol. Chem.53,1433, (1989) biotransformation Phytochemistry, 40, 1149, (1995)section U.S. Pat. No. 42346684 Alkylation of any N or EP-49981 Epoxidesdescribed in EP-49981. O with epoxide Alkylation of any N or O withalkyl-leaving group, i.e iodide, bromide, mesylate, tosylate etc.EP-49981

Subtitution of C- leaving group with alcohol or amine CH-648-326-A

Reductive alkylation of N EP-49981

Reductive amination of carbonyl EP-240175-A

Addition to carbonyl with organometallic species Tetrahedron, Vol51,No.33, 9063-9078, (1995), Bull. Soc. Chim. Fr.,134, 777-784, (1997)

Oxidation of alcohol Synlett, (5), 617-619, (1999) Org. Lett, 1 (9),1475-1478 (1999) Acylation. Note X = suitable leaving group, iechloride, organic acid etc. U.S. Pat. No. 4,175,123

C—C double bond formation from carbonyl or lactol Tetrahedron Assymetry3(3), 451-8 (1992). J.Org. Chem. 61(11), 3594-3598, (1996)

All the substances mentioned herein can be labelled e.g. with isotopesof certain atoms, as is well known in the art. Suchisotopically-labelled substances are available by well-known methods inthe art.

Preferred inhibitors include acarbose and higher homologues thereof,Trestatin A, Trestatin C, the compound of Fraction 21 of Example 7below, Example 8 below, as well as the fermentation broth productsmentioned below.

A preferred group of inhibitors are substantially pure single compound,or partially-purified fermentation or biotransformation product,inhibitors including acarbose and higher homologues thereof, TrestatinA, Trestatin C, the compound of Fraction 21 of Example 7 below, Example8 below.

Especially preferred are acarbose and Trestatin C.

Some of the inhibitors may be made by biotransformation/fermentation,such as the methods described herein below.

Biotransformation/fermentation Products

The cultures Streptomyces conglobatus ATCC31005, Streptomyces coelicolorsubsp. flavus ATCC19894, Streptomyces kursannovii ATCC 11912 andStreptomyces lienomycini ATCC43687 were obtained from the American TypeCulture Collection (ATCC located at 10801 University Boulevard,Manassas, Va. 20110-2209, U.S.A.). The cultures Streptomyces sp. KC672isolated from a marine sediment in Suruga Bay, Japan and Streptomycessp. CL45763 have been deposited in accordance with the Budapest Treatyat the National Collections of Industrial and Marine Bacteria Ltd. andassigned the accession numbers NCIMB41058 and NCIMB41057 respectively.(NCIMB is located at 23 St. Machar Drive, Aberdeen, U.K. AB24 3RY.). Thedepositor was Pfizer Central Research, Pfizer Limited, Ramsgate Road,Sandwich, Kent, CT13 9NJ, United Kingdom. Pfizer Ltd. is a wholly-ownedsubsidiary of Pfizer Inc. 235 East 42nd Street, New York, N.Y., USA.

In addition, mutant strains of Streptomyces conglobatus ATCC31005,Streptomyces coelicolor subsp. flavus ATCC19894, Streptomyceskursannovii ATCC 11912, Streptomyces lienomycini ATCC43687, Streptomycessp. KC672 and Streptomyces sp. CL45763 can be used. Such mutant strainscan be obtained spontaneously, or by the application of knowntechniques, such as exposure to ionising radiation, ultraviolet light,and/or chemical mutagens such as N-methyl-N-nitrosourethane,N-methyl-N′-nitro-N-nitrosoguanadine, ethyl methyl sulphate etc.Genetically transformed and recombinant forms include mutants andgenetic variants produced by genetic engineering techniques, includingfor example recombination, transformation, transduction, protoplastfusion etc.

Fermentation of the cultures of Streptomyces conglobatus ATCC31005,Streptomyces coelicolor subsp. flavus ATCC19894, Streptomyceskursannovii ATCC11912, Streptomyces lienomycini ATCC43687, Streptomycessp. KC672 and Streptomyces sp.CL45763 can be carried out using standardprocedures well known in the art for filamentous bacteria of the genusStreptomyces. For example growth of the organism may take place onsuitable solid medium or aqueous liquid medium under aerobic conditionsin the range 24 to 35° C. using suitable sources of carbon, nitrogen andtrace elements such as iron, zinc, manganese for 2 to 30 days.

Use is made of the following fermentation media. AP5-H Production MediumCorn starch (Hidex) 80 g Yeast extract (Oxoid) 5 g K₂HPO₄ 1 g MgSO₄.7H₂O1 g Glutamic acid 1 g FeSO₄.7H₂O 0.01 g ZnSO₄.2H₂O 0.001 g MnSO₄.2H₂O0.001 g CaCO₃ 7 g Tap water 1 l NaOH To pH 7.0 ½ strength MECO MediumGlucose 5 g Acid Hydrolysed Starch (Hidex ™, Japan) 10 g Casitone ™(Difco - nitrogen source) 2.5 g Yeast extract (Oxoid ™) 2.5 g Wheatembryo (Sigma) 2.5 g Calcium carbonate 2.0 g Demineralised water 1 lNaOH To pH 7.0 Modified ANG-3 medium Soluble starch 20 g Glucose 100 gSoya Flour (Trusoy ™) 10 g NaNO₃ 2 g K₂HPO₄ 1 g MgSO₄.7H₂O 0.5 g KCl 0.5g FeSO₄.7H₂O 0.01 g MOPS buffer (Sigma) 20 g Demin water 1 l NaOH to pH7

All the media described can be supplemented with other starches,partially hydrolysed starches or soluble starches and or sugars such asD-xylose, D-ribose, D-maltose, D-maltotriose, D-sedoheptulose,D-trehalose, D-glucose and or nitrogen sources such as asparagine,aspartate and glutamine.

EXAMPLE 1 Preparation of a Fermentation Broth Demonstrating Rumen Fluidα-amylase Inhibitory Activity from Streptomyces conglobatus ATCC31005

Streptomyces conglobatus ATCC31005 maintained on a ¼ strength ATCC172agar slope was inoculated as a loopful of spores into two 300 mlErlenmeyer flasks each containing 50 mls of ½ strength MECO medium. Theywere then allowed to incubate for 7 days at 28° C., 200 rpm on an InforsMultitron Shaker with 1″ (2.5 cm) throw. At this point the broth wascentrifuged at 3500 rpm and the supernatant removed from the mycelium.The α-amylase inhibitory activity was determined for the supernatantwhich is summarised in the table below.

Supernatant dilution into assay 1:1000 1:10000 1:100000 % inhibitionflask 1 91 89 65 % inhibition flask 2 86 90 58

EXAMPLE 2 Preparation of a Fermentation Broth Demonstrating Rumen Fluidα-amylase Inhibitory Activity from Streptomyces sp. CL 45763

Streptomyces sp. CL45763 maintained on an agar slope of Bacto ISP-3 wasinoculated as a loopful of spores into four 300 ml Erlenmeyer flaskseach containing 50 mls of AP5-H medium. They were then allowed toincubate for 9 days at 28° C., 200 rpm on an Infors Multitron Shakerwith 1″ (2.5 cm) throw. At this point the broths were combined,centrifuged at 3500 rpm, and then the supernatant removed from themycelium. The α-amylase inhibitory activity for the supernatant was thendetermined which is summarised in the table below.

Supernatant dilution into assay 1:1000 1:10000 1:100000 % inhibition 7977 53

EXAMPLE 3 Preparation of a Fermentation Broth Demonstrating Rumen Fluidα-amylase Inhibitory Activity from Streptomyces coelicolor subsp. flavusATCC19894

Streptomyces coelicolor subsp. flavus ATCC19894 maintained on a ¼strength ATCC172 agar slope was inoculated as a loopful of spores intoten 300 ml Erlenmeyer flasks each containing 50 mls of AP5-H medium.They were then allowed to incubate for 4 days at 28° C., 200 rpm on anInfors Multitron Shaker with 1″ (2.5 cm) throw. At this point the brothswere centrifuged at 3500 rpm and the supernatant removed from themycelium. The α-amylase inhibitory activity was determined for thecombined supernatants which is summarised in the table below.

Supernatant dilution into assay 1:1000 1:10000 % inhibition flask 1 6635

EXAMPLE 4 Preparation of a Fermentation Broth Demonstrating Rumen Fluidα-amylase Inhibitory Activity from Streptomyces kursannovii ATCC11912

Streptomyces kursannovii ATCC11912 maintained on a ¼ strength ATCC172agar slope was inoculated as a loopful of spores into two 300 mlErlenmeyer flasks each containing 50 mls of AP5-H medium. They were thenallowed to incubate for 5 days at 28° C., 200 rpm on an Infors MultitronShaker with 1″ (2.5 cm) throw. At this point the broths were centrifugedat 3500 rpm and the supernatant removed from the mycelium. The α-amylaseinhibitory activity was determined for each supernatant which issummarised in the table below.

Supernatant dilution into assay 1:1000 1:10000 1:100000 % inhibitionFlask 1 85 73 27 % inhibition Flask 2 85 70 30

EXAMPLE 5 Preparation of a Fermentation Broth Demonstrating Rumen Fluidα-amylase Inhibitory Activity from Streptomyces lienomycini ATCC43687

Streptomyces lienomycini ATCC43687 maintained on a ¼ strength ATCC172agar slope was inoculated as a loopful of spores into three 300 mlErlenmeyer flasks each containing 50 mls of modified ANG-3 medium. Theywere then allowed to incubate for 5 days at 28° C., 200 rpm on an InforsMultitron Shaker with 1″ (2.5 cm) throw. At this point the broths werecentrifuged at 3500 rpm and the supernatant removed from the mycelium.The α-amylase inhibitory activity was determined for each individualsupernatant which is summarised in the table below.

Supernatant dilution into assay 1:1000 1:10000 1:100000 % inhibitionflask 1 87 70 41 % inhibition flask 2 85 75 41 % inhibition flask 3 8576 47

EXAMPLE 6 Preparation of a Fermentation Broth Demonstrating Rumen Fluidα-amylase Inhibitory Activity from Streptomyces sp. KC672

Streptomyces sp. KC672 maintained on a ¼ strength ATCC172 agar slope wasinoculated as a loopful of spores into two 300 ml Erlenmeyer flasks eachcontaining 50 mls of AP5-H medium. They were then allowed to incubatefor 7 days at 28° C., 200 rpm on an Infors Multitron Shaker with 1″ (2.5cm) throw. At this point the broths were centrifuged at 3500 rpm and thesupernatant removed from the mycelium. The α-amylase inhibitory activitywas determined for the supernatants which is summarised in the tablebelow.

Supernatant dilution into assay 1:1000 1:10000 % inhibition flask 1 5421 % inhibition flask 1 49 22

EXAMPLE 7 Isolation of a Rumen Fluid α-amylase Inhibitor fromStreptomyces conglobatus ATCC31005

A loopful of spores of Streptomyces conglobatus ATCC31005 maintained on¼ strength ATCC172 agar was inoculated into two 300 ml Erlenmeyerflasks, each containing 50 ml of AP5-H medium. After 24 hours incubationat 28° C., 200 rpm on an Infors Multitron Shaker with 1″ (2.5 cm) throw,these flasks were used to inoculate two 5 liter minijars (Electrolab™,Gloucester, U.K) each containing 3.5 liters each of AP5-H medium. Thesebroths were incubated at 28° C. with an aeration of 3 l/min and stirringat 300 rpm for 6 days. At harvest the broths were centrifuged at 2500rpm and the supernatants decanted. They were each stirred twice for 45minutes with 86 g of charcoal and then filtered through a 500 g bed ofArbocel™. The pH at this stage was pH7. Each carbon cake was extractedtwice with 860 mls of aqueous acetone (1:1) maintaining the pH between 2and 3 by the addition of concentrated hydrochloric acid. The fouraqueous acetone extracts were partially evaporated, combined andlyophilised to give 40 g of a brown solid. This solid was then dissolvedin 500 mls of deionised water and applied to a column of 750 mlsAmberlite IR120 (H⁺ form) at a flow rate of 2 ml/min. The column wasthen washed with 1 l. of water and then eluted with 5N ammonia solutioncollecting 50 ml fractions. Fractions 13 to 28 were lyophilised to give1.4 g of a dark brown powder. This was then dissolved in 15 mls ofwater, filtered and the resulting filtrate diluted with 5 mls ofacetonitrile. This was injected in 2 ml volumes on to a Cosmosil NH₂-MScolumn (20×250 mm) and eluted at 20 mls/min with acetonitrile water(60:40). Fractions were collected every 30 seconds and analysed by LC-MSusing a Finnegan AQA™ instrument. Fractions containing M+H⁺970 werecombined and dried down to give a gum solid, 70 mgs. The solid was thendissolved in two mls of water, filtered and injected in two halves on toan Waters Aqua™ 5 micron 125A column (21×150 mm), using a Waters DeltaPrep™ 4000 system with diode array detection. Fractions were collectedand two, both fraction 21, were combined containing the M+H⁺970 peakresulting in a white solid, 6.5 mg.

Accurate mass data was collected on a Bruker Apex II FT-ICR-MS 4.7 Tinstrument where the sample, dissolved in methanol/water/acetic acid(50:50:1) at approx 0.5 mgs/ml, was introduced into an Analyticaelectrospray source by direct infusion at 4 μl/min.

m/z (ESI, FTMS) [M+H]⁺=970.3601, C₃₇H₆₄NO₂₈ requires 970.3609

m/z (ESI, FTMS) [M+Na]⁺=992.3452, C₃₇H₆₃NO₂₈Na requires 992.3429

The NMR (proton, carbon-13, TOCSY, HSQC and HMBC) and mass spectra ofthis fraction 21 compound, also known as “6942/99/1” are consistent withthe structure shown below.

The compound shown above has been disclosed in GB patent 1 482 543 and(Ag. Biol. Chem. 46(7) (1982) 1941).

Table: ¹H and ¹³C NMR chemical shifts of 6942/99/1 (δ in ppm relative tointernal dioxane)

Position δ_(H) multiplicity J (Hz) δ_(C) A1-CH₂OH 4.09/4.20 2 × d 14.264.5 A1 — — — 141.9 A2 5.87 d, br 5.3 126.6 A3 3.51 t, br ˜5 58.9 A43.63 m — 73.9 A5 3.73 m — 75.9 A6 4.01 d, br 6.9 74.2 B1 5.28 m 3.6102.8 B2 3.57 m — 75.6 B3 3.58 m — B4 2.45 t 9.7 67.8 B5 3.72 m — 72.6B6-Me 1.31 d 6.4 20.2 Rings C-E: 1 ˜5.37 d 4.0 ˜102.4 2 ˜3.60 dd 9.5,4.0 ˜74.4 3 ˜3.93 t ˜9.5 ˜76.2 4 ˜3.62 t ˜9.5 ˜79.9 5 ˜3.81 m — ˜74.26-CH₂ ˜3.79/3.83 m — ˜63.4 α-F1 5.20 d 3.9 94.9 α-F2 3.54 dd ˜9.5, 3.9α-F3 3.94 t ˜9.5 76.2 α-F4 3.62 t ˜9.5 ˜79.9 α-F5 3.91 m — 72.8 α-F6-CH₂˜3.8 m — ˜63.4 β-F1 4.63 d 7.9 98.8 β-F2 3.25 dd 7.9, 9.5 76.9 β-F3 3.74t 9.5 79.1 β-F4 3.63 dd 9.5, 8.3 ˜79.9 β-F5 3.56 m — 77.5 β-F6-CH₂ ˜3.87m — ˜63.4

The “Fraction 21 compound” was found to have an inhibiting effect in theamylase screen mentioned herein.

EXAMPLE 8 Isolation of a Rumen Fluid α-amylase Inhibitor fromStreptomyces conglobatus ATCC-31005

The AP5-H Production Medium mentioned above was used as fermentationmedium.

Streptomyces conglobatus ATCC31005 maintained on a ¼ strength ATCC172agar slope was inoculated as a loopful of spores into two 300 mlErlenmeyer flasks each containing 50 mls of AP5-H medium. They were thenallowed to incubate for 24 hours at 28° C., 200 rpm on an InforsMultitron Shaker with 1″ throw. At this point the inoculum wastransferred into a 3 liter Fernbach flask containing 1 liter of AP5-Hmedium and incubated for a further 24 hours under the same conditions asdescribed for the Erlenmeyer flasks. This inoculum was then transferredto 20 liters of AP-5H medium which had previously been sterilised in a30 liter New Brunswick Micros™ stainless steel fermenter. The broth wasthen agitated at 300 rpm at 28° C. with 20 l/min air for 112 hours andthen harvested.

The harvested broth was centrifuged using a Carr Powerfuge™ at 20 000G.To the supernatant, at a natural pH of 7.8, was added 500 g of activateddecolourising charcoal (Aldrich 16155-1) and the mixture stirred for 16hours. Following filtration through a filter aid, such as Arbocel™, thesupernatant was treated with a further 500 g charcoal, for 1 hour in thesame manner. The combined charcoal cakes were washed with aqueousmethanol (10 L 1:1) and then extracted twice with aqueous acetone (10 L1:1) by stirring for 1 hour followed by filtration through filter aid.Following partial rotary evaporation and freeze drying, 98.7 g ofbiologically active material were obtained.

This material was dissolved in 1800 ml demineralised water and loadedonto a column of 3.5 L Amberlite IR 120(H)™ at a rate of 5 ml/minute.Following a water wash (2 L) the product was eluted with 1 L aliquots of5N ammonia solution. Following freeze drying, the most potent fractions(5 to 8) were combined to give 10.8 g of brown solid.

1.1 g of this material was purified by chromatography, in five equalinjections, using a Waters Delta Prep 4000™ chromatography system, a250×21.2 mm CromasilNH₂ (ex Phenomenex) and a gradient from 67%acetonitrile 33% water to 50/50 at 20 minutes at a flow rate of 24ml/minute. 12 ml fractions were collected.

The fractions containing the peak of interest (31 to 35 from each run)were combined to give 138 mg white solid. This material waschromatographed again, this time using a 150×21.2 mm Aqua column (exPhenomenex) and a gradient from 100% water to 90% water 10% acetonitrileover 15 minutes at a flow rate of 21.2 ml/minute. Fractions werecollected at half minute intervals. A total of 43 mg of desired product,I was obtained from fractions 22 and 23.

The observed data are consistent with the following structure, referredto herein as the “Example 8 compound”:

M/z (ESI, FT-MS) [M+H]⁺=1435.546 corresponding to a molecular formula ofC₅₆H₉₅N₂O₄₀ (+/−3.028 ppm).

M/z (ESI, FT-MS) [M+Na]⁺=1457.528 corresponding to a molecular formulaof C₅₆H₉₄N₂O₄₀Na (+/−1.914 ppm).

All NMR data given below were recorded on a Varian Innova 600 MHzmachine at 10° C. in D₂O using a 3 mm probe.

Position H Multiplicity J (Hz) C A1-CH2OH 4.09/4.21 2 × d 14.2 64.2 A1 —— — 141.7 A2 5.88 d, br 5.3 126.2 A3 3.52 t, br ˜5 58.8 A4 3.64 m —˜73.9 A5 3.74 m — ˜75.9 A6 4.01 d, br 7.2 ˜73.7 B1 5.33 m 3.6 ˜102.2 B23.57 m — 75.5 B3 3.58 m — ? B4 2.45 t 9.7 67.7 B5 3.72 m — 72.2 B5-Me1.31 d 6.4 20.0 C1 5.36 d 3.8 100.1 C2 3.59 dd — 73.8 C3 3.89 t — 76.2C4 3.61 t — ˜79.3 C5 3.88 m — ˜74.2 C6-CH2 3.82 m — ˜63.1 D1-CH2OH4.09/4.21 2 × d 14.1 64.7 D1 — — — 139.1 D2 5.95 d, br 4.1 128.9 D3 3.52t, br ˜5 57.7 D4 3.82 m — 71.9 D5 4.15 dd 8.2, 5.3 73.3 D6 4.21 d, br6.9 78.4 E1 5.33 m 3.6 ˜102.2 E2 3.57 m — 75.6 E3 3.58 m — ? E4 2.45 t9.7 66.7 E5 3.72 m — 72.2 E5-Me 1.31 d 6.4 20.0 Rings F-H:1 ˜5.40 d 3.8˜102.2 2 ˜3.59 dd 9.5/d.0 ˜75.2 3 ˜3.93 t ˜9.5 ˜76.1 4 ˜3.65 m ˜9.5˜79.1 5 ˜3.82 m — ˜73.8 6-CH2 ˜3.79/3.83 m — ˜63.1 α-l1 5.21 d 3.8 94.9α-l2 3.54 dd ˜9.5, 3.9 α-l3 3.94 t ˜9.5 76.2 α-l4 3.62 t ˜9.5 ˜79.9 α-l53.91 m — 72.8 α-l6-CH2 ˜3.8 m — ˜63.4 β-l1 4.63 d 7.9 98.7 β-l2 3.25 dd7.9, 9.5 76.9 β-l3 3.74 t 9.5 79.1 β-l4 3.63 dd 9.5, 8.3 ˜79.9 β-l5 3.56m — 77.5 β-l6-CH2 ˜3.87 m — ˜63.4

NB where a “?” appears in the table above, there was severe signaloverlap meaning that an unambiguous assignment could not be made

Modifications to Acarbose and Other Related α-amylase and/orα-glucosidase Inhibitors

Biotransformations

Microbial Biotransformation

Microbial whole organisms capable of glycosylation of acarbose or otherrelated α-amylase and/or α-glucosidase inhibitors could be used to giveincreased α-amylase and/or α-glucosidase inhibitory activity, whichinclude Bacillus subtilis ATCC55060¹ , Saccharopolyspora erythraeATCC11635² and a blocked mutant of S.avermitilis ATCC53567³. Otherorganisms which can glucosidate include Cunninghamella sp. NRRL5695¹⁴and Beauvaria bassiana DSM 875 and DSM 1344¹⁵. Moreover the microbialdirected biosynthesis of acarbose by an Actinoplanes sp. CBS 793.96⁴ fedwith rutin could also be used with other related α-amylase and/orα-glucosidase inhibitor producing organisms. These may give analogues ofacarbose or related acarbose like homologues which could alsodemonstrate increased α-amylase and/or α-glucosidase inhibitoryactivity.

Moreover microorganisms capable of O-acylation¹⁶, oxidation (incl.epoxidation¹⁷ and ketone¹⁸ formation), hydroxymethylation¹⁹O-methylation²⁰, etc. can also be used to make new analogues of acarboseand related analogues which could also demonstrate increased α-amylaseand/or α-glucosidase inhibitory activity.

Crude, Partially Purified and Purified Enzyme Biotransformations

Enzymatic methods of glycosylation can be used to synthesise or modifyoligosaccharides. Specific protection and deprotection of hydroxylgroups is not required and the enzymes only transfer to one or twohydroxyl groups. This often leads to fewer reaction steps and simplerpurification procedures.

Transglycosylation of Bacillus stearothermophilus maltogenic amylase(BSMA) with acarbose and various acceptors have been used, where theenzyme was an Escherichia coli transformant carrying the BSMA gene.⁵Here it was observed that the BSMA cleaved the first glycosidic bond ofacarbose to give the pseudotrisaccharide (PTS) and then added on aglucose unit at the α (1→6) position to give isoacarbose, where acarboseitself has an α (1→4) linkage at the terminal glucose. The addition of anumber of different carbohydrates to the digest gave transfer productsin which the PTS was primarily attached a (1→6) to D-glucose, D-mannose,D-galactose and methyl α-D-glucopyranoside. With D-fructopyranose andD-xylopyranose, PTS was linked at α (1→45) and α (1→4) respectively. α-αTrehalose and maltitol both gave two major products with PTS linked α(1→6) and α (1→4) to the glucopyranose residue. PTS was primarilytransferred to C-6 of the nonreducing residue of maltose, cellobiose,lactose and gentiobiose. Sucrose gave PTS linked α (1→4) to the glucoseresidue. Raffinose gave two major products with PTS linked α (1→6) and α(1→4) to the D-galactopyranose residue. Maltotriose gave two majorproducts with PTS linked α (1→6) and α (1→4) to the nonreducing endglucopyranose residue. Xylitol gave PTS linked α (1→5) as the majorproduct and D-glucitol gave PTS linked α (1→6) as the only product. Allthese examples may show improved α amylase inhibitory activity.

Other groups of enzymes can be used to produce glycosidated analogues ofacarbose or other amylase and/or α-glucosidase inhibitors that haveaccessible sugar or hydroxyl groups. Enzymatic preparations that may beused include α- and β-galactosidase, α- and β-mannosidase,β-N-acetylglucosaminidase, β-N-acetylgalactoaminidase, and α-Lfucosidase.⁶ Glycosidation can take place at either end of thevalienamine or cyclitol unit of acarbose and experience shows that theglycosyl transfer is preferred to take place at the non reducingterminal monosaccharide unit of substrates.⁷ Studies using endoglycosidases may lead to branched structures. The enzyme preparationsdescribed can be microbially derived e.g Aspergillus niger, A. terreus,A. oryzae, Bacillus circulans, B. stearothermophilus, Coccobacillus, orinsect juice e.g snail, or plant derived e.g apples, mushrooms, alfalfaseeds, defatted almond meal etc.⁶

Glycosyltransferases can also be used to glycosylate acarbose andrelated analogues demonstrating α-amylase inhibitory activity but aremuch rarer enzymes.⁸ Many of these glycosyltransferases have beencloned. They are often referred to as being rather stringent to thedistal one to two saccharide moieties and are also very specific to theglycosyl donor. They can be persuaded to work with both unnatural donorsand/or acceptors maintaining their advantages of strict regio andstereoselectivity and high yields. Only a few glycosyltransferases arereadily available and most experiments have been carried out withgalactosyltransferase Gal T.⁹

A special group of glycosyl transferases are cyclodextringlucanotransferases (CGTase). These enzymes are produced bymicroorganisms and many are commercially available. They catalysecyclodextrination of starch but also transfer one or more α-glucosylunits to various acceptors. They can be used for extending glycosides orfor α-glucosylation of many compounds. CGTase from B. stearothermophiluswas used for the transglucosylation of rutin where the glucosyl unit wasextended by one or more glucose units.¹⁰ A similar approach could beused for acarbose and related α-amylase and/or α-glucosidase inhibitorscontaining glucose units.

Another approach is to use glycogen phosphorylase which is the wellknown enzyme responsible for the formation or degradation of α (1→4)glucans. Phosphorylase requires an activated substrate such as theglucosyl phosphate ester. With this substrate a glucan chain, the primerunit, can be elongated by glucose units with the release of phosphate.¹¹

Modification of acarbose and related α amylase and/or α-glucosidaseinhibitors containing sugar units can also be made using selectivehydrolyses with α amylase itself which can either cleave sugar units ortransglycosylate^(12,13)

For other modifications of the hydroxyl groups of sugar units acylases,esterases, lipases, hydrolases and dehydratases can also be used.

REFERENCES FOR THIS SECTION

-   1. Petuch B. R et al. Microbial transformation of immunosuppressive    compounds 111. Glucosylation of immunomycin and FK506 by Bacillus    subtilis ATCC55060. J. Ind. Microbiol. 13, 131-135, (1994)-   2. Arison, B. H et al. Microbial glycosidation of avermectins. Eur.    Pat. Appl. (1992) EP 520557 A1.-   3. Pacey M. S. et al. Preparation of 13-epi-selamectin by    biotransformation using a blocked mutant of Streptomyces    avermitilis. J. Antibiotics, 53 (3), 301-305, (2000)-   4. Cruegar, A et al. Novel acarviosin glycoside: synthesis of a new    saccharase inhibitor via biotransformation. Ger. Offen. (1999) DE    19821038 A1-   5. Park, H. P et al. Transglycosylation reactions of Bacillus    stearothermophilus maltogenic amylase with acarbose and various    acceptors. Carbohydrate Res. 313, (1998), 235-246-   6. M. Scigelova et al. Glycosidases—a great synthetic tool. J. of    Molecular Catalysis B: Enzymatic 6, (1999), 483-494.-   7. Crout D H G, et al. Glycosidases and glycosyltransferases in    glycoside and oligosaccharide synthesis. Curr. Opin. Chem. Biol    2(1),(1998), 98-111,-   8. Kren, V., et al. Glycosylation employing bio-systems: from    enzymes to whole cells. Chem. Soc. Rev. 26, (1997), 463-   9. C. H. Wong. et al Enzymes in Synthetic Organic Chemistry.    Tetrahedron Org. Chem. Ser. Eds. Baldwin J. E & Magnus P. D.,    Pergamon (1994), Vol 12.-   10. Suzuki et al. Agric. Biol. chem. Enzymatic formation of    4-α-D-glucopyranosyl-rutin 55, (1991), 181-   11. Evers B et al. Further syntheses employing phosphorylase.    Bioorganic & Medicinal Chemistry 5(5), (1997), 857-863-   12. Takada, M et al. Chemo-enzymic synthesis of    galactosylmaltooligosaccharidonolactone as a substrate analogue    inhibitor for mammalian α-amylase. Japan. J. Biochem. (Tokyo)    123(3), (1998), 508-515-   13. Kim, T. K et al. Synthesis of glucosyl-sugar alcohols using    glycosyltransferases and structural identification of    glucosyl-maltitol. J. Microbiol. Biotechnol. 7(5), (1997),310-317.-   14. Chatterjj, P et al. Glucosidation of betulinic acid by    Cunninghamella sp. J. Nat. Prod. 62(5), (1999), 761-763.-   15. Kittleman, M et al. Microbial hydroxylation and simultaneous    formation of the 4″-O-methylglucoside of the tyrosine-kinase    inhibitor CGP 62706. Chimia 53(12), (1999), 594-596.-   16. Oda S et al. Double coupling of acetyl coenzyme A production and    microbial esterfication with alcohol acetyltransferase in an    interface bioreactor. J. Ferment. Bioeng. 83, (1997), 423-428-   17. Garcia-Granados et al. Biotransformation of ent-6α-acetoxy- and    ent-6-ketomanoyl oxides with Rhizopus nigricans ATCC10404 and    Curvularia lunata ATCC12017. Phytochemistry 45, (1997),283-291.-   18. Fantin, G et al. Regioselective microbial oxidation of bile    acids. Tetrahedron, 54, (1998), 1937-1942-   19. Azerad, R. Patent application WO 99/47963 dated 23, Sep. 1999.    Novel method for the production of fexofenadine using Absidia    corymbifera LCP 63-1800 or Streptomyces platensis NRRL 2364.-   20. Sariaslani, F et al. Novel biotransformations of    7-ethoxycoumarin by Streptomyces griseus. NRRL8090. Appl. Environ.    Microbiol.46(2), (1983), 468-474.

Certain of the substances mentioned herein can exist in one or moregeometric and/or stereoisomeric forms. The present disclosure includesall such individual isomers and salts and prodrugs thereof. Certaincompounds mentioned herein could exist in more than one tautomeric form.Similarly certain compounds mentioned herein may have zwitterionicforms. It is to be understood that the disclosure embraces all suchtautomers, zwitterions and their derivatives.

The disclosure includes veterinarily acceptable salts of the compoundsmentioned herein, including the acid addition and the base salts thereofwhere appropriate. Suitable acid addition salts are formed from acidswhich form non-toxic salts and examples are the hydrochloride,hydrobromide, hydroiodide, sulphate, hydrogen sulphate, nitrate,phosphate, hydrogen phosphate, acetate, maleate, fumarate, lactate,tartrate, citrate, gluconate, succinate, benzoate, methanesulphonate,benzenesulphonate and p-toluenesulphonate salts. Suitable base salts areformed from bases which form non-toxic salts and examples are thealuminium, calcium, lithium, magnesium, potassium, sodium, zinc anddiethanolamine salts. For a review on suitable salts see Berge et al, J.Pharm. Sci., 66, 1-19 (1977).

It will be appreciated by those skilled in the art that certainprotected derivatives of compounds mentioned herein, which may be madeprior to a final deprotection stage, may not possess the desiredbiological activity as such, but may, in certain instances, betransformed after administration into the body, for example bymetabolism, to form compounds mentioned herein which are biologicallyactive. Such derivatives are included in the term “prodrug”. It willfurther be appreciated by those skilled in the art that certain moietiesknown to those skilled in the art as “pro-moieties”, for example asdescribed in “Design of Prodrugs” by H Bundgaard (Elsevier) 1985, may beplaced on appropriate functionalities when such functionalities arepresent in compounds mentioned herein, also to form a “prodrug”.Further, certain compounds mentioned herein may act as prodrugs of othercompounds mentioned herein. All protected derivatives, and prodrugs, ofthe compounds mentioned herein are included within the scope of thedisclosure.

The skilled person will appreciate that certain substances mentionedherein can be made by methods other than those hereinafter described, byadaptation of the methods herein described and/or adaptation of methodsknown in the art, for example the art described herein, or usingstandard textbooks such as

“Comprehensive Organic Transformation—A Guide to Functional GroupTransformations”, R C Larock, VCH (1989 or later editions),

“Advanced Organic Chemistry—Reactions, Mechanisms and Structure”, J.March, Wiley-Interscience (3rd or later editions),

“Organic Synthesis—The Disconnection Approach”, S Warren (Wiley), (1982or later editions),

“Designing Organic Syntheses”, S Warren (Wiley), (1983 or latereditions), “Guidebook To Organic Synthesis” R K Mackie and D M Smith(Longman) (1982 or later editions),

“Methoden der Organischen Chemie”, Houben Weyl, Georg Thieme Verlag,Stuttgart,

“The Chemistry of the Hydroxyl Group” Parts 1 & 2, Saul Patal, (1971),Interscience Publishers,

“The Chemistry of the Amino Group”, Saul Patal, (1968), IntersciencePublishers,

“Trends in Synthetic Carbohydrate Chemistry”, ACS Symposium Series 386,(1989), American Chemical Society, Washington, D.C.,

“Advances in Carbohydrate Chemistry and Biochemistry”, Volumes 1-39,Academic Press, New York

“Carbohydrate Chemistry”, Volumes 1-11, The Chemical Society, London,

“Methods in Carbohydrate Chemistry”, Volumes 1-8, Academic Press, NewYork,

“Carbohydrates Synthetic Methods and Applications in MedicinalChemistry”, Ogura, H. et al, (1982), Kodansha, Tokyo. etc.

and the references therein as a guide.

It is to be understood that the synthetic transformation methodsmentioned herein are exemplary only and they may be carried out invarious different sequences in order that the desired compounds can beefficiently assembled. The skilled chemist will exercise his judgmentand skill as to the most efficient sequence of reactions for synthesisof a given target compound. For example, substituents may be added toand/or chemical transformations performed upon, different intermediatesto those mentioned hereinafter in conjunction with a particularreaction. This will depend inter alia on factor such as the nature ofother functional groups presents in a particular substrate, theavailability of key intermediates and the protecting group strategy (ifany) to be adopted. Clearly, the type chemistry involved will influencethe choice of reagent that is used in the said synthetic steps, theneed, and type, of protecting groups that are employed, and the sequencefor accomplishing the synthesis. The procedures may be adapted asappropriate to the reactants, reagents and other reaction parameters ina manner that will be evident to the skilled person by reference tostandard textbooks and to the examples provided hereinafter.

It will be apparent to those skilled in the art that sensitivefunctional groups may need to be protected and deprotected duringsynthesis of a compound of the invention. This may be achieved byconventional methods, for example as described in “Protective Groups inOrganic Synthesis” by T W Greene and P G M Wuts, John Wiley & Sons Inc(1999), and references therein. Functional groups which may desirable toprotect include oxo, hydroxy, amino and carboxylic acid. Suitableprotecting groups for oxo include acetals, ketals (e.g. ethylene ketals)and dithianes. Suitable protecting groups for hydroxy includetrialkylsilyl and diarylalkylsilyl groups (e.g. tert-butyldimethylsilyl,tert-butyldiphenylsilyl or trimethylsilyl) and tetrahydropyranyl.Suitable protecting groups for amino include tert-butyloxycarbonyl,9-fluorenylmethoxycarbonyl or benzyloxycarbonyl. Suitable protectinggroups for carboxylic acid include C₁₋₆ alkyl or benzyl esters.

The amylase and/or α-glucosidase inhibitors may be administered eitheralone or in combination with one or more agents used in the treatment(including prophylaxis) of disease or in the reduction or suppression ofsymptoms as appropriate for the treatment of acidosis and relatedconditions. Examples of such agents (which are provided by way ofillustration and should not be construed as limiting) include buffers,antibiotics including ionophores, probiotics, organic acids andbacteriocins, antiparasitics, eg fipronil, lufenuron, imidacloprid,avermectins (eg abamectin, ivermectin, doramectin), milbemycins,organophosphates, pyrethroids; antihistamines, eg chlorpheniramine,trimeprazine, diphenhydramine, doxylamine; antifungals, eg fluconazole,ketoconazole, itraconazole, griseofulvin, amphotericin B;antibacterials, eg enroflaxacin, marbofloxacin, ampicillin, amoxycillin;anti-inflammatories eg prednisolone, betamethasone, dexamethasone,carprofen, ketoprofen; dietary supplements, eg gamma-linoleic acid; andemollients.

The amylase and/or α-glucosidase inhibitors can be administered alonebut will generally be administered in admixture with a suitableexcipient, diluent or carrier selected with regard to the intended routeof administration and standard pharmaceutical/veterinary/farmingpractice.

Advantageously for treatment of livestock animals such as sheep andcattle, the active agent can be administered orally using suitablestandard methods such as mixed with the animal's feedstuff, in thedrinking fluid or via a bolus delivered directly to the rumen. Forin-feed administration a concentrated feed additive or premix may beprovided for mixing with the normal animal feed. Additional physical andchemical stabilising agents may also be included to maintain or enhancethe stability of the active agents in the said formulation.

The methods by which the active agent may be administered include oraladministration by capsule, bolus, tablet or drench, or, alternatively,they can be administered by injection or as an implant into the rumen.Such formulations may be prepared in a conventional manner in accordancewith standard veterinary practice.

For example, the active agent can be administered orally in the form ofsolutions, powders or suspensions, which may contain flavouring orcolouring agents, for immediate-, delayed-, modified-, sustained-,pulsed- or controlled-release applications.

In addition to in-feed or in-drink administration with part of thecattle's normal diet, it is envisaged that the active agent could beseparately administered between normal feeding and drinking, e.g. in theform of a palatable “treat” such as in a molasses-based formulation.

For aqueous suspensions and/or elixirs, the active agent may be combinedwith various sweetening or flavouring agents, colouring matter or dyes,with emulsifying and/or suspending agents and with diluents such aswater, ethanol, propylene glycol and glycerin, and combinations thereof.Additional physical and chemical stabilising agents may also be includedto maintain or enhance the stability of the active agents in the saidformulation.

The active agent may also be delivered via a long-acting bolusformulation directly to the rumen, wherein the formulation device isretained within the ruminoreticular sac for prolonged periods of time tofacilitate sustained release. Ruminal retention of the formulationdevice as described in this instance may be achieved using densematrices or reservoirs based on aluminium or steel cylinders or pelletsformed from a mixture of clay, drug and other ingredients.

The active agent may, in certain cases, also be administeredparenterally, for example, intravenously, intra-arteriallyintraperitoneally, intramuscularly or subcutaneously, or administered byinfusion techniques. For such parenteral administration the active agentis best used in the form of a sterile aqueous solution which may containother substances, for example, enough salts or glucose to make thesolution isotonic with blood. The aqueous solution should be suitablybuffered (preferably to a pH of from 3 to 9), if necessary. Thepreparation of suitable sterile parenteral formulations is readilyaccomplished by terminal sterilisation methodology or by asepticmanufacture using standard pharmaceutical techniques well known to thoseskilled in the art.

Thus unit doses of the active agent may contain from 0.001 mg to 20 g ofactive agent for administration singly or two or more at a time, asappropriate. For example acarbose has been administered at 15 g peranimal per day in 2 separate feeds. A target range for an activecompound is up to ca. 3 g/animal/day. The vet/farmer in any event willdetermine the actual dosage that will be most suitable for anyindividual animal or group of animals and it may vary with the age,weight, diet and response of the particular animal. The above dosagesare exemplary of the average case. There can, of course, be individualinstances where higher or lower dosage ranges are merited and such arewithin the scope of this invention. The skilled person will appreciatethat, in the treatment of certain conditions such as acute acidosis theactive agent may be given as a single dose as needed or desired.

The active agent will normally be administered orally or by any othersuitable route (which can eventually reach the rumen), in the form ofpreparations comprising the active ingredient, optionally in the form ofa non-toxic organic, or inorganic, acid, or base, addition salt, in anacceptable veterinary/pharmaceutical dosage form. Depending upon thedisorder and animal to be treated, as well as the route ofadministration, the compositions may be administered at varying doses(see below).

While it is possible to administer the active agent directly without anyformulation, the active agents are preferably employed in the form of apharmaceutical, or veterinary, formulation comprising apharmaceutically, or veterinarily, acceptable carrier, diluent orexcipient and active agent. The carrier, diluent or excipient may beselected with due regard to the intended route of administration andstandard pharmaceutical, and/or veterinary, practice. Compositionscomprising the active agent may contain from 0.1 percent by weight to90.0 percent by weight of the active ingredient.

The formulations will vary with regard to the weight of active compoundcontained therein, depending on the species of animal to be treated, theseverity and type of condition and the body weight of the animal. Forparenteral and oral administration, typical dose ranges of the activeingredient are 0.0001 to 1000 mg per kg of body weight of the animal.Preferably the range is 0.001 to 20 mg per kg. For example acarbose wasadministered at 16 mg/kg. More preferably the range is 0.001 to 5 mg/kg,and most preferably 0.001 to 0.5 mg/kg.

It is to be appreciated that all references herein to treatment includecurative, palliative and prophylactic treatment.

The efficacy of agents can be demonstrated using the following TestMethods, in which acarbose is used as an example of a suitable amylaseand/or α-glucosidase inhibitor.

TEST METHODS

Rumen Bacterial Amylase Assay—Protocol 1

The assay utilises a Sigma amylase kit (577) to determine whethercompounds inhibit the action of rumen fluid supernatant amylases. Theenzymatic reactions involved in the assay are as follows:5 ET-G₇PNP→(α-amylase)→2 ET-G₅+2 G₂PNP+2 ET-G₄+2 G₃PNP+ET-G₃+G₄-PNP2G₂PNP+2 G₃ PNP→(α-glucosidase)→4 PNP+10 glucose

α-Amylase hydrolyses 4,6-ethylidene-G₇-PNP (ET-G₇PNP) to G₂, G₃ and G₄PNP fragments. α-Glucosidase (α-1,4-glucan glucohydrolase EC 3.2.1.3)hydrolyses G₂PNP and G₃PNP to yield p-nitrophenol and glucose. Fivemoles of substrate (ET-G₇PNP) is hydrolysed to yield 4 moles ofp-nitrophenol. p-Nitrophenol absorbs light as 405 nm, and following atwo minute lag period the rate of increase in absorbance at 405 nm isdirectly proportional to α-amylase activity in the well.

Rumen fluid was collected from 4-month-old Hereford x Friesian calves(125-135 kg, supplied by Cwmnant Calves Ltd. Cwmnant. Tregaron.Ceredigion) fed on diet GH 313. The rumen fluid was collected fromslaughtered calves into pre-warmed vacuum flasks, as soon as possibleafter euthanasia. It was then filtered through a double layer ofabsorbent gauze (Absorbent gauze BP, GAUZ 4 from Robert Bailey plc,Stockport) to remove hay and feed particles. The liquid was centrifugedat a relative centrifugal force of 23,300 for 60 minutes, and thesupernatant decanted, avoiding contamination from the loose top layer ofthe pellet by careful pouring. The supernatant was then aliquoted into50 ml plastic tubes and frozen at −20° C. When required for use in anassay the rumen fluid supernatant was thawed by standing the tubes incold water. Test compounds or controls were dispensed into the 96-wellassay plate at 4 μl/well. 100 μl per well of Sigma amylase reagent 577(made up to half the volume described in the instructions (i.e. 10 mlfor a 577-20 vial)) was then added to each well, followed by 100 μl perwell of rumen fluid supernatant. A T=0 reading at 405 nm was taken atthis stage using an Anthos plate reader. The plate was then incubated atroom temperature or 37° C. until an optical density window ofapproximately 1.000 U was seen (typically one hour at 37° C. or threehours at room temperature). A second reading was taken at 405 nm, andthe first reading subtracted from it. Active compounds cause a reductionin the optical density readings when compared to the control without theagent being tested.

Results - IC50s in rumen fluid amylase screen (using Sigma kit 577)Number of Compound Average IC50 (μM) assays Acarbose 2.03 n = 34Trestatin A 0.17 n = 8 Trestatin B 2.89 n = 6 Trestatin C 0.09 n = 6V-1532 0.57 n = 2 Example 7 2.06 n = 8 Example 8 0.44 n = 10

Dose Response of Acarbose in the Rumen Bacterial Amylase Assay [AcarboseConcentration in Molar Units]

Rumen Fluid Glucosidase Assay Protocol

This assay is used to determine IC50 values for inhibitors of bacterialglucosidase activity from bovine rumen fluid cell suspension (RFCS)using a colorimetric assay.

The assay measures conversion of maltose into glucose. Rumen fluid cellsare incubated with maltose in the presence of inhibitors, and the amountof glucose produced is assessed using a red colourimetric endpoint. Thehigher the level of inhibition the lower the glucose produced and theless red colour produced. The plates are read at 450 nm.

Main reaction:

-   -   Maltose+glucosidase→glucose

Glucose assay

-   -   Glucose+ATP→G-6-P and ADP (Hexokinase and Mg²⁺)    -   G-6-P and NADP→6-phosphogluconate (6-PG) and NADPH    -   NADPH+phenazine methosulfate (PMS)→NADP+PMSH    -   PMSH+INT (iodonitrotetrazolium chloride)→PMS+INTH

INTH is deep red coloured.

Rumen fluid was collected from a fistulated five year old dry Guernseydonor cow fed twice daily on 1.4 kg GH313 and 2.3 kg hay. The rumenfluid was collected into pre-warmed vacuum flasks. It was then filteredthrough a double layer of absorbent gauze (Absorbent gauze BP, GAUZ 4from Robert Bailey plc, Stockport) to remove hay and feed particles. Theliquid was centrifuged at a relative centrifugal force of 650 for 15minutes to remove food particles and protozoa. The supernatant wasdecanted into fresh tubes and centrifuged at a relative centrifugalforce of 23,300 for 60 minutes. The supernatant and the loose top layerof the pellet were discarded. The pellet was resuspended in PBS at 1:8of original volume i.e. 50 ml of PBS for the pellet from 400 ml of rumenfluid, and frozen at −20° C. When required for use the cells were thawedby standing the tube in cold water, and then diluted to 4.5 μl ofcells/well (45 μl/ml).

Test compounds or controls were dispensed into the 96-well assay plateat 2 μl/well, followed by 50 μl per well of 10 mM maltose and 50 μ/wellof rumen fluid cell suspension. The plate was incubated at 37° C. for 1hour. Sigma glucose detection kit 115A was reconstituted by addition of17 ml of Millipore water and 4 ml of colour reagent to each vial. 100 μlof this solution was added per well and the plate returned to the 37° C.incubator for 45 minutes. The plate was then read at 450 nm. Activecompounds cause a reduction in the optical density readings whencompared to the no-inhibitor control wells.

Results - IC₅₀s in rumen fluid glucosidase screen Compound Average IC50(μM) Number of assays Acarbose 1.08 n = 7 Trestatin A 33.6 n = 3Trestatin B 4.10 n = 2 Trestatin C 148.5 n = 2 V-1532 65.5 n = 2 Example7 6.38 n = 1 Example 8 13.1 n = 2

Rumen Bacterial Amylase Assay Protocol 2

The assay utilises digestion of amylose covalently linked to RemazolBrilliant Blue R to determine whether compounds inhibit the action ofrumen fluid supernatant amylases. When the insoluble substrate isincubated with amylase blue dye is released into the well. This can bemeasured spectrophotometrically to determine how much amylase activityis present, and whether test compounds are inhibitors of amylase.

Rumen fluid was collected from a fistulated five year old dry Guernseydonor cow fed twice daily on 1.4 kg GH313 and 2.3 kg hay. The rumenfluid was collected into pre-warmed vacuum flasks. It was then filteredthrough a double layer of absorbent gauze (Absorbent gauze BP, GAUZ 4from Robert Bailey plc, Stockport) to remove hay and feed particles. Theliquid was centrifuged at a relative centrifugal force of 23,300 for 60minutes, and the supernatant decanted, avoiding contamination from theloose top layer of the pellet by careful pouring. The supernatant wasthen aliquoted into 50 ml plastic tubes and frozen at −20° C. Whenrequired for use in an assay the rumen fluid supernatant was thawed bystanding the tubes in cold water. 100 μl per well of a 2% suspension ofamylose azure (Sigma A3508) was added to each well from a beaker thatwas stirred throughout to ensure an even distribution of substrate. Testcompounds or controls were dispensed into the 96-well assay plate at 4μl/well, followed by 100 μl per well of rumen fluid supernatant. Theplate was then incubated at 37° C. for 2.25 hours. 100 μl of liquid wasremoved gently from each well using a 12-channel pipette, transferred toa fresh 96-well plate and read at 620 nm. Active compounds cause areduction in the optical density readings when compared to theno-inhibitor control wells.

Results - IC50s in rumen fluid amylase screen (amylose azure) CompoundAverage IC50 (μM) Number of assays Acarbose 2.39 n = 4 Trestatin A 0.79n = 2 V-1532 2.07 n = 2 Example 7 0.56 n = 2 Example 8 9.45 n = 2

Protocol for Determination of Minimum Inhibitory Concentrations (MICs)in Aerobes

MICs were determined by a standard agar dilution technique according tothe National Committee for Clinical Laboratory Standards (NCCLS, M7Edition A2). An outline of the method employed is detailed below.

The MICs were determined using the standard test medium, Mueller Hinton(MH) agar (Unipath).

Preparation of agar plates: 19 ml of test medium was added toappropriate doubling dilutions of test compound (1 ml) and mixedthoroughly. The mixture was poured into a petri dish (90 mm) and theagar allowed to solidify.

Preparation of inoculum: Four to five colonies of the test organism wereinoculated from a MH agar plate culture into 10 ml MH broth (Unipath).The broth was incubated at 37° C. until visibly turbid. The density ofthe culture was adjusted to a turbidity equivalent to that of a 0.5McFarland standard by the addition of saline (0.85% v/v).

Inoculation of agar plates: The plates were dried for approximately 1hour in a 37° C. incubator. Plates were inoculated with a MultipointInoculator (Denley). The pins on this device deliver 0.001 ml inoculumto the plate (equivalent to 10⁴-10⁵ organisms).

Incubation of plates: Plates were inverted and incubated at 37° C. for18 hours.

Determination of endpoints: MICs were recorded as the lowestconcentration of test compound that completely inhibited growth,disregarding a single colony or a faint haze caused by the inoculum.

References for this Section

National Committee for Clinical Laboratory Standards

Methods for dilution antimicrobial susceptibility tests for bacteriathat grow aerobically—second edition.

Approved standard reference methods for the determination of MIC ofaerobic bacteria by broth macrodilution, broth microdilution and agardilution. Chair holder J. Allan Waitz, PhD DNAX Research Institute,NCCLS Document M7-A2

Villanova, Pa.: NCCLS, 1990

RESULTS USING ACARBOSE IN THIS TEST: No. Bacterial species MIC (μg/ml) 1E. coli >128 ATCC 10418 2 E448 >128 3 E454 >128 4 E459 >128 5 E450 >1286 E476 >128 7 E461 >128 8 E516 >128 9 E517 >128 10 E520 >128 11 Salm.enteritidis >128 B1234 12 B1227 >128 13 B1240 >128 14 B1218 >128 15B1231 >128 16 B1233 >128 17 B1232 >128 18 B1235 >128 19 E. faecium >1281.1.7 20 1.2.4 >128 21 1.1.6 >128 22 28.7.7 >128 23 1.2.6 >128 2428.6.7 >128 25 5.4 >128 26 4.5 >128 27 3.1 >128 28 10.1  >128 29 E.faecalis >128 1.3.10 30 1.4.12 >128 31 1.1.16 >128 32 1.6.6 >128 331.3.13 >128 34 1.10.4 >128 35 28.5.7 >128 36 1.9.5 >128 37 1.1.4 >128 3828.6.9 >128 39 S. aureus >128 3.3 40 3.4 >128 41 3.5 >128 42 5.1 >128 436.1 >128 44 8.2 >128 45 8.3 >128 46 9.3 >128 47 10.3  >128 48 10.4  >12849 NCTC >128 6571

DIETS USED IN THE FOLLOWING TESTS

[All diets were provided by Grain Harvesters Ltd, The Old Colliery,Wingham, Canterbury, Kent CT3 1LS, England.]

Material Inclusion Analysis GH313: BARLEY (fine) 24.000 VOLUME 100.000WHEAT 10.000 PROTEIN 14.005 WHEAT MIDDLINGS 11.900 OIL 3.794 SUNFLOWERMEAL (EXT) 5.100 FIBRE 8.501 RAPESEED MEAL (EXT) 10.000 STARCH 27.309PEAS 7.500 STARCH + SUGAR 32.783 WHOLE LINSEED 1.200 GRAIN SCREENINGS7.500 UNMOLASSED SUGAR BEET 13.900 LIMESTONE GRANULES 1.300 SALT 0.800GHS CATTLE SUPP. 0.250 ADDAROME Cattle Supplement 0.020 MOLASSES 5.000VEGETABLE FAT (MIXER) 1.500 99.970 GH633 BARLEY (fine) 22.100 VOLUME100.000 WHEAT MIDDLINGS 17.500 PROTEIN 15.122 MAIZE GLUTEN 8.800 OIL4.700 FISHMEAL (PROVIMI 66) 2.500 FIBRE 9.601 SUNFLOWER MEAL (EXT) 4.500STARCH 17.175 RAPESEED MEAL (EXT 00) 5.000 SUGAR 8.770 LUCERNE PELLETS10.000 MOLASSED SUGARBEET 20.000 LIMESTONE FLOUR 0.400 SALT 0.650 INTLAMB SUPPLEMENT (10 kg) 1.000 SPRAY VEGETABLE FAT 1.600 MOLASSES 5.000MIXER VEGETABLE FAT 1.000 100.050 GH651: BARLEY (fine) 15.000 VOLUME100.000 WHEAT 50.000 PROTEIN 13.984 WHEAT MIDDLINGS 11.000 OIL 3.206RAPESEED MEAL (EXT) 14.400 FIBRE 4.539 LIMESTONE GRANULES 1.900 STARCH39.523 DICALCIUM PHOSPHATE 0.030 STARCH + SUGAR 44.838 SALT 0.820 GHSCATTLE SUPPLEMENT 0.250 COLBORN No. 3 0.100 MOLASSES 5.000 VEG FAT(MIXER) 1.500 100.000 GH654: WHEAT MIDDLINGS 22.000 VOLUME 100.000 MAIZEGLUTEN 16.600 PROTEIN 12.867 SUNFLOWER MEAL (EXT) 7.000 OIL 4.688RAPESEED MEAL (EXT) 4.200 FIBRE 13.991 OATFEED 10.000 STARCH 10.109 N.I. Straw 10.000 SUGAR 5.352 UNMOLASSED SUGAR BEET 20.000 LIMESTONEGRANULES 1.200 SALT 0.680 CALCINED MAGNESITE 0.540 AMMONIUM CHLORIDE0.050 COLBORN Cattle Supplement 0.250 ADDAROME Cattle Supplement 0.040MOLASSES 5.000 VEG. FAT (MIXER) 2.500 100.060

Evaluation of Agents Using the Rumen Simulation Technique (RUSITEC) toModel Chronic Acidosis.

The in vitro rumen simulation technique (RUSITEC), first described byCzerkawski and Breckenridge (1977) was used to evaluate the effect ofthe bacterial α-amylase and/or α-glucosidase inhibitor acarbose on dailypH profiles and VFA production using a commercial cattle concentrateration (GH313—see later). Feeding 30 g/d of this ration with 2.5 g/dchopped barley straw had previously been found to give total volatilefatty acid (VFA) concentrations of more than 150 mM i.e. concentrationsassociated with chronic acidosis in vivo (Nagaraja, Galyean & Cole,1998, supra)

Equipment: The apparatus consisted of two RUSITEC units each containingfour-fermentation vessels. Each vessel had a volume of 1 liter, and washeated to 39° C. in a water bath. The feed was placed in a nylon bag(14×9 cm, 50 μm), and was gently agitated using a piston mechanism (8strokes/min). Buffer (McDougall, 1948) was continuously infused at arate of approximately 750 ml/day by an eight channel peristaltic pump(Watson Marlow). The effluent was collected in 1 liter glass bottlescontaining 20 ml of oxalic acid solution (12 g/100 ml in deionisedwater). This was added to inhibit further microbial activity

Feed: Each vessel was fed daily with a bag containing 30 g of thecommercial pelleted ration GH313 (89% dry matter) and 2.5 g barley straw(90% dry matter) chopped into 1-2 cm lengths. 7 g of corn starch (SigmaCat. No. S4126) was added to the liquid phase of all fermenter vesselsat feeding on the last four days of the experiment to simulate acuteacidosis.

Rumen fluid donor: Rumen fluid was collected from a five year old dryGuernsey cow. The animal was fed twice daily with 1.4 kg GH313 and 2.3kg hay. Rumen contents were collected via a rumen fistula (Bar DiamondInc. P.O. Box. 60. Bar Diamond Lane. Parma. Id. 83660-0060. U.S.A)

Vessel inoculation: Rumen contents were taken from the fistulated donoranimal at 08:00 h (before the morning feed). The material was carried tothe laboratory in pre-warmed insulated flasks, and then strained throughfour layers of cotton gauze into another pre-warmed insulated flask. 70g of the solid residue were weighed into each of eight nylon bags. Onebag containing rumen solids, and one bag containing fresh feed wasplaced in the feed chamber for each vessel. The liquid contents of eachvessel were 100 ml deionised water, 200 ml of buffer artificial and 500ml of rumen fluid. After assembling and sealing the vessels, they wereplaced in the water baths and the piston rod attached to the drive bar.The effluent tubes were placed in the collection flasks. The head spacein each vessel was flushed with CO₂ for 2 min., then the piston drivemotor and buffer infusion pump were started.

Daily maintenance and sampling procedure: These procedures were carriedout at the same time each day. Eight feed bags were prepared, and a 1 ldispenser bottle containing infusion buffer warmed to 39° C.

-   1. Drive motors were switched off and the infusion pump stopped.    Infusion lines clamped and disconnected from the pump.-   2. The fermentation vessels were removed from the water bath and    serviced in turn.-   3. For each vessel, the feed chamber was extracted and feed bags    exchanged. On Day 2 the new bag replaced the one containing rumen    solids, whilst on subsequent days the new bag replaced the one that    has been incubated for 48 hours. Chamber then replaced in    fermentation vessel.-   4. The removed bag was placed in a small plastic bag and 25 ml    buffer added from the dispenser. The bag was washed by squeezing in    the buffer for 20 seconds, then the liquid was poured into the    vessel. This washing procedure was repeated twice with fresh buffer.-   5. After reassembling the vessel, it was replaced in the water bath    and attached to the drive bar. The buffer line was reconnected and    the pH electrode relocated. The effluent collection bottle was    exchanged and the vessel headspace purged with CO₂ whilst the next    vessel was being serviced.-   6. This process was repeated for all vessels, then the drive motors    and infusion pump were restarted when gassing was complete. For the    last vessel gassing was for a similar duration as for the other    vessels.

Treatment with acarbose: Acarbose was obtained as Glucobay™ tablets(Bayer, AAH Pharmaceuticals) Each tablet contains 100 mg acarbose.Duplicate vessels were treated with 0, 1, 10 and 100 mg/d of acarbose byadding 1 tablet to each of two feed bags to give 100 mg/vessel/d. Thelower doses were prepared by dissolving/suspending a tablet in 10 mlbuffer (giving a 10 mg/ml solution of acarbose). One ml of this solutionwas then added to 9 ml of buffer, giving a 1 mg/ml solution. One ml ofeach solution was then added to the contents of two feed bags, to give10 and 1 mg/vessel/d. Finally, the acarbose was dried on to the feed byleaving the bags at room temperature overnight.

Analyses: Dry matter losses from the nylon bags after 48 hoursincubation were measured by drying the washed bag contents in an ovenfor 23 h at 65° C. Effluent samples (10 ml) were taken daily and storedat −20° C. for subsequent VFA and lactate analysis. pH was automaticallyrecorded at 17 min. intervals using equipment supplied by Philip HarrisEducation. A combination electrode was fitted in each vessel via agas-tight port in the lid. Each electrode was connected to aSensorMeter, and four SensorMeters were connected to one DL plus 128datalogger. The recorded pH values were downloaded to a PC runningDatadisk 32 software (Philip Harris Education) and then transferred to aspreadsheet for further analysis. The electrodes were removed from thevessels (when the feed bags were being changed), rinsed and placed inpH7.0 standard buffer. The reading were 7.0+/−0.1 units throughout theexperiment. The electrodes were recalibrated to pH 7.0 before beingreplaced in the vessels. VFAs were measured by adding 0.1 ml of asolution containing a mixture of 25 g/100 ml metaphosphoric acid and 1.2g/100 ml crotonic acid to 1 ml of effluent. This mixture was centrifugedfor 10 min. at 12000 g and an aliquot of the supernatant transferred toan autosampler vial. VFAs were resolved and quantified on a HewlettPackard 6890 series gas chromatograph fitted with an autosampler andflame ionisation detector. The acids were resolved on a SGE Ltd 25 meterBP21 column (0.33 mm O.D., 0.22 mm I.D. 0.25 um film thickness).Nitrogen was used as the carrier gas with a flow rate of 1.9 ml/min. Theoven temperature was 165° C., and injection ports and detectors wereheld at 250° C. Concentrations were calculated by using crotonic acid asan internal standard, and the system was calibrated using a standardsolution containing acetic, propionic, butyric, iso-valeric andn-valeric acids. L-lactic acid was measured using Sigma kit 826, andD-lactate was measured using the same procedure, except L-LDH wasreplaced with D-LDH (Sigma Catalogue No. L2011) and L-lactate wasreplaced with D-lactate (Sigma Catalogue No. L0625). Assays was carriedout on a 96-well microtitre plate and the absorbances measured using anAnthos microtitre plate reader fitted with a 340 nm filter. The systemwas calibrated by preparing solutions of D- and L-lactate from 0 to 100mM.

Schedule:

Day

0 Inoculate.

3 Start collecting effluent

5 Start daily pH measurement.

10 Begin dosing with acarbose (0, 1, 10 or 100 mg/vessel/d) to pairs ofvessels.

Continue to end of experiment.

18 Add extra starch to all vessels.

22 End experiment.

REFERENCES FOR THIS SECTION

Czerkawski, J. W. and Breckenridge, G. 1977. Design and development of along-term rumen simulation technique (RUSITEC). British Journal ofNutrition, 38, 371-384.

McDougall, E. I. 1948. Studies on ruminant saliva. 1. The compositionand output of sheep's saliva. Biochemical Journal, 43, 99-109.

Rusitec Results: Acarbose

Comparing the treatment period with the preceding control periodindicated a dose-related change in VFA production of −16%, −10% +3% and−3% in response to additions of 100, 10, 1 and 0 mg/vessel/d ofacarbose. There was a general shift of fermentation products fromacetate and propionate to butyrate with all treatments between thecontrol and treatment periods, resulting in increases in butyrateproduction of 134%, 76%, 27% and 24% respectively for the above doses.There was no L-lactate accumulation in this study, confirming that themodel represented chronic rather than acute acidosis.

RUSITEC Result Using Example 8 and Acarbose

Experimental Outline:

Daily maintenance procedures as previously described for Acarbose.

Rumen fluid donor cow—Fed GH313 pellets plus barley straw.

Daily Feed—30 g GH313 pellets plus 2.5 g chopped barley straw.

Treatment Preparation

Acarbose: 1 tablet added to each bag to give 100 mg/vessel/d

EXAMPLE 8

Eight×57 mg pre-weighed samples stored in fridge in 380 2.1. On the daybefore the feed bags were to be placed in RUSITEC, a 57 mg sample wasdissolved in 2.28 ml buffer (25 mg/ml). 0.21 ml of this solution wasadded to 1.9 ml buffer to give a 2.5 mg/ml solution. 1 ml of eachsolution was added to a prepared feed bag to give 25 and 2.5mg/vessel/d. and dried overnight at room temperature.

Schedule:

Day

0 Inoculate.

4 Start collecting effluent

5 Start daily pH measurement

9 Review data, allocate vessels to treatments.

10 Begin treatments.

18 End experiment.

Results and Conclusions: In this experiment 0.02 mM Example 8 gave anincrease in mean daily pH of 0.3 units compared to 0.7 units for 0.2 mMacarbose. Treatments of 100 mg acarbose, 25 or 2.5 mg Example 8 pervessel per day, or none (control) caused changes in total VFA productionof −15%, −8%, −1% and −11% when compared with the preceding controlperiod. There was a trend for redistribution of fermentation products,with butyrate production increasing in the treatment period comparedwith the control. The proportional increases were 113%, 20%, 18% and 1%respectively. There was no L-lactate accumulation in this study,confirming that the model represented chronic rather than acuteacidosis.

In Vitro Rumen Propionic Acid Screen

Reagents

Rumen Fluid: An eight year old dry Guernsey cow, fitted with a rumenfistula (Bar Diamond Inc. P.O. Box. 60. Bar Diamond Lane. Parma. Id.83660-0060. U.S.A) was fed twice daily with 1.4 kg GH633 and 2.3 kg hay.This animal was used as a source of rumen contents, which were taken at08:00 h (before the morning feed). The material was carried to thelaboratory in a pre-warmed insulated flask, and then strained throughfour layers of cotton gauze into another pre-warmed bottle, which wasstored in an incubator at 40° C. until the fluid was dispensed into theassay tubes.

Buffer: Dissolve the following in deionised water. g/l g/500 mlNa₂HPO₄.2H₂O 9.88 4.94 KH₂PO₄ 3.40 1.70 NaH₂PO₄.H₂O 1.11 0.55 Adjust topH 7.0 with 1 M NaOH.

Deoxygenate by bubbling with an oxygen-free gas mixture (10% CO₂, 5% H₂in nitrogen) for at least 5 min.

Substrate Mixture:

-   -   68 g corn starch. Ex Sigma Cat. S-4126.    -   17 g α-cellulose. Ex Sigma Cat. C-6429.    -   15 g Type 1 soya flour. Ex Sigma Cat. S-9633.    -   Mix well.

Immediately before use, suspend in buffer at 200 mg/ml.

Metaphosphoric/crotonic Acids Solution:

The following were dissolved in deionised water.

-   -   25% (w/v) metaphosphoric acid. Ex. BDH Cat. 291904A plus    -   1.2% (w/v) crotonic acid. Ex Sigma Cat. C-4630

VFA Standard Mixture:

The following were dissolved in 100 ml of deionised water.

concen- nominal tration mwt weight (mg) mM Sodium acetate. Ex Sigma Cat.S-7670 136.1 680 50 Sodium propionate. Ex Sigma Cat. P-1880 96.1 192 20Sodium butyrate. Ex Sigma Cat. B-5887 110.1 110 10 n-Valeric acid. ExAldrich Cat. 24,037-0 102.1 102 10 iso-valeric acid. Ex Sigma Cat.I-7128 102.1 102 10

1 ml of metaphosphoric/crotonic acids solution added to 10 ml of VFAmixture then aliquoted into automatic liquid sampler vials.

Procedure:

The assay was conducted in 16 ml Sorvall centrifuge tubes

A tablet containing 100 mg of acarbose was placed in 10 ml of buffer andshaken until the tablet was completely disrupted, giving a 10 mg/mlsolution of acarbose. This solution was serially diluted to 2, 0.4, 0.08and 0.016 mg/ml with buffer.

One ml of these solutions was added to triplicate assay tubes (givingfinal assay mixture concentrations of 1000, 200, 40, 8 and 1.6 ug/ml).Control tubes were prepared by replacing the acarbose solution withbuffer. One ml of substrate suspension was added to all the assay tubes,followed by 3 ml of warmed degassed buffer and then by 5 ml of strainedrumen liquor. Suba-Seal stoppers (No. 29) were fitted, and the headpressure in the tubes reduced by passing a hypodermic needle attached toa vacuum line through the stopper until the tube contents frothed. Thetubes were then placed in a 40° C. incubator for 6 hours and shakenhourly.

Pre-incubation VFA concentration were determined by preparing threetubes as for incubation but 1 ml metaphosphoric/crotonic acids solutionwas added immediately following the rumen liquor. These tubes werestored at 4° C. and processed with the post-incubation tubes.

The incubation was terminated after 6 h by removing the stoppers andadding 1 ml of metaphosphoric/crotonic acids solution. The tubes werethen centrifuged for 8 minutes at 18,000 g at 4° C., and an aliquot ofthe supernatant stored in an automatic liquid sampler vial untilrequired for VFA analysis by gas chromatography. (as described inRUSITEC protocol)

Result Calculation:

Production of total VFA and propionate during the incubation wasdetermined as follows. Firstly, first the pre-incubation concentrationsof total VFA and propionic acid were calculated as the mean of theanalyses of the pre-incubation samples. Then for each incubated sample,post- minus pre-incubation concentration gave production duringincubation. The molar proportion of propionic acid in total VFA.producedduring the incubation was also calculated.

The total VFA and % propionic acid values were meaned for the replicatetubes, and the mean control total VFA and % propionic acid valuesnormalised as 100%, then the change caused by the test treatmentsexpressed relative to this value.

acarbose *Dose μg/ml Total VFA % Propionate 1000 50 88 200 58 81 40 6374 8 72 74 1.6 92 94 0.32 96 97 0 100 100

In vivo Testing in Fistulated Cattle

Objective: To determine the effect of the agent for testing, in thiscase acarbose, on chronic rumen acidosis induced in fistulated cattle. Arumen pH profile representative of chronic acidosis was induced bystepwise increase in the level of concentrate feeding of a specifieddiet and a reduction in the roughage offered. This was followed bytreatment of each animal with acarbose, administered via a permanentrumen fistula, to assess its ability to normalise rumen pH.

Experimental Animals: Six fistulated Hereford x Friesian steers,weighing 170-230 kgs (supplied by Cwmnant Calves Ltd. Cwmnant, Tregaron,Ceredigion)

Treatment: Glucobay® 100. Acarbose 100 mgs per tablet.

Management: The cattle were fed GH651 cattle high cereal beef pellets(variable amount) with barley straw (variable amount) divided over twoequal feeds, given at around 08.00 hr and 14.30 hr each day. Precisefeeding times were recorded. Water was available ad-lib. Cattle wereindividually housed in pens (9 square meters per pen) in a buildingenvironmentally controlled to 16° C.

Design:

Acarbose Volume No. of Group Treatment Form^(n) Route (g/trt) (ml)Animals 1 Acarbose Aq. Sol. Through 4.0 100 6 fistula

While chronic acidosis was being induced, manual pH measurements weretaken approximately 5 and 8 hours post-morning feed. Rumen fluid sampleswere taken for VFA and lactate analysis. Once a suitable pH profile wasgenerated (see procedure section), each steer was fitted with a harnessto carry an automated pH sampling and recording device (Philip HarrisPlus 128 Data logger.+p.H. First Sense Recorder). Rumen pH values wereautomatically recorded every 17 minutes for a maximum of 21 days. Rumenfluid samples (10 ml) were taken twice daily for measurement of VFAlevels, molar ratios and lactate levels. These were collected (bymanually removing a sample of rumen content with a small stainless steelladle, filtering and transferring to a 10 ml polypropylene vial) justbefore acarbose was added to the rumen at both dosing times. The pHprobes were removed for cleaning and recalibration immediatelypre-morning feed.

Results: The daily pH curves were used to calculate the period thatrumen pH was below pH5.5, and therefore indicative of chronic acidosis.

Rumen fluid samples were taken at 13:00 and 16:00 i.e. before and afterthe afternoon feed. There was little difference in total VFAconcentrations at the 13:00 samples, but the VFA concentration in the14:00 samples fell during the treatment period. This was consistent withthe pH profiles. At all sampling times the percentage of propionate waslower during the treatment period. There was no accumulation of lacticacid in the rumen fluid samples, indicating that the animals did notexperience acute acidosis during the experiment.

EFFICACY OF ACARBOSE IN LACTATING DAIRY CATTLE

The study measured the effect of acarbose on rumen pH, milk yield andmilk composition in lactating cows in which chronic acidosis had beeninduced by offering a highly fermentable diet. The study includedmeasurement of rate of adaptation to the introduction and removal ofacarbose.

Experimental animals were six lactating multiparous Holstein/Friesiancows between 5 and 11 years of age and 500-750 kg, with permanent rumencannulas. The animals started the experiment in early lactation, but notbefore peak lactation, to allow greater experimental sensitivity. Theprincipal measurements in this study were pH in the ventral sac of therumen, milk yield and milk composition.

Management practices complied with the UK Home Office code of practicefor the Housing and Care of Animals Used in Scientific Procedures(1989).

Design: Animals enrolled into the study received test article for 21consecutive days from the start time points described below.

Treatment Supplement fed Supplement fed Supplement fed Number of Groupfrom Day 0 from Day 21 from Day 42 Animals T01 A B A 3 T02 B A B 3 A =Control supplement ration B = Acarbose containing supplement ration

Procedure:

Masking/Bias-Reducing Methods: Six lactating multiparousHolstein/Friesian cows were enrolled on to the study on Day—1. Animalswere paired based on their calving date (cows with similar dates pairedtogether). Within each pair, one animal received T01 and the other T02.The treatment was assigned at random. Where possible, the randomisationwas constrained so that the average feed intakes per treatment groupwere similar.

Methods: At approximately two weeks prior to Day 0, eight animals begana preliminary feeding period designed to identify a feeding regimen thatinduced acidotic pH levels in the rumen. Each cow was held in a tiestall from this point until completion of the study. Control total mixedration (TMR) was fed and where necessary the amount and compositionadjusted to establish a minimum rumen pH of 5.0-5.5. The mean intakeover the last 5 days of the preliminary period was calculated and thismean amount was offered to each animal throughout the trial. Unconsumedfood was removed and weighed on a daily basis and prior to the morningfeed.

The TMR was supplemented with 0.5 kg/day ground wheat with either noadditive (Control) or containing the test article (acarbose at 15 g perday), and offered separately in equal halves over the morning andafternoon feeds. An automatic watering system was also used to offerwater ad-libitum.

TMR composition: Ingredient % in total ration DM Grass silage 10.0 Maizesilage 30.0 Cracked wheat 16.7 Ground barley 9.2 Rapeseed meal 4.1Soyabean meal 6.1 Molassed SBP 9.2 Wheatfeed 8.2 Regumaize 4.0 Fishmeal1.0 Minerals 1.5 Total 100.0

From the start of the study, all animals were milked twice daily througha pipeline system at approximately 06.30 h and 16.00 h. Milk yieldweight was manually recorded and then transcribed to an electronic dailymilk file.

On Day—1, each animal was physically examined by a veterinary surgeon toassess physical and clinical normality. Animals met all of the inclusioncriteria and none of the exclusion criteria.

On Day 0, the test article feeding regimen described in DESIGN began.Three animals followed the design Control/Treatment/Control (A/B/A) onthree consecutive experimental periods whilst a second group of threeanimals followed the opposite treatment sequence (B/A/B). Experimentalfeeding periods lasted 3 weeks, consisting of 2 weeks for adaptation anda final week for detailed measurements.

Animals were fed twice daily at unequal intervals at approximately 08.00h and 15.00 h (i.e. 7 and 17-h intervals) to allow rumen sampling for pHmeasurement to be concentrated during the period of minimum pH(estimated to be 2-4 h after the second feed). Each animal was offered0.5 kg/day ground wheat supplement to the TMR divided equally over thetwo feeds and containing either no additive (Supplement A) or the testarticle (Supplement B).

Each animal completed the study after the final pH measurement of thethird experimental period on Day 62.

Measurements: Automated pH measuring equipment (Philip Harris Plus 128Data logger+pH First Sense Recorder) was used to record rumen pH valuesevery 17 minutes. A 10 mL-15 mL sample of rumen fluid taken at 07.30 hrsand two samples taken at approximately minimum pH (at approximately18.00 hrs and 20.00 hrs) were frozen immediately at −20° C. for furtheranalysis. Fresh weight of TMR offered and refused was recorded daily foreach animal. During non-measurement weeks (i.e. prelim period andadaptation weeks 1 and 2 in each experimental period), dry matter (DM)of main forage components (grass and maize silages) were determinedapproximately weekly (or more frequently if it appears necessary fromvisual assessment of the silage). For measurement weeks (i.e. week 3 ineach experimental period), the DM of the TMR were measured daily for thelast 5 days while a bulk of each of the individual TMR components (grasssilage, maize silage, concentrate mix, ground wheat) were prepared fromthe same 5 days for subsequent diet analysis (i.e. one sample of eachmain feed per period). Only one sample of Regumaize was taken during thestudy as it is a single bulk liquid. Refusals were sampled for DMdetermination during the last 5 days of each experimental period. Single20 ml milk samples for fat, protein and lactose were taken at am and pmmilkings on 3 alternate days in each adaptation week. During measurementweek milk samples were taken on the last 5 consecutive days. Each milksample was analysed separately. Additionally a further 20 ml milk samplewas taken on each occasion that milk was sampled during the measurementweeks and immediately frozen at approximately −20° C. for possiblesubsequent analysis. Daily milk yield data was generated by totallingthe morning and afternoon milkings on a given date. Live weight wasmeasured in each experimental period for each animal.

Results: Rumen pH was calculated as time below pH5.5 (i.e. in a state ofchronic acidosis) for the treatment and control periods. The averagetime below pH 5.5 was 4.3 hours for control periods and 3.4 hours fortreatment periods. Milk fat increased dramatically in treated animals,from an average of 1033 grams per day to 1281 grams per day. Theproportion of fat in the milk was also increased, from 32.8 g/L to 46.1g/L.

Acute Acidosis Model: Assessment of Acarbose

Experimental Design:

Treatment No. Description Route No. Animals 1 Control: ˜200 mL water BIDfor Cannula 5 (3 dry cows and 7 days pre-challenge, 12.5 g/kg BW 2heifers) of challenge mixture* 2 Acarbose: 1.07 mg acarbose/kg BWCannula 5 (2 dry cows and dissolved in ˜200 mL water BID for 3 heifers)7 days pre-challenge, 12.5 g/kg BW of challenge mixture containing .02g/kg BW acarbose *48.4% cornstarch, 48.4% ground corn, 2.1% sodiumcaseinate, 1.1% urea (food grade) suspended in approximately 5 gallonslukewarm water

Procedures: Ten Holstein dried off cows and heifers (initial weight740±27 (SE) kg, range=606-870 kg) were group-housed during thepre-treatment period and individually housed during the experimentalperiod. Animals were moved to a head-gate during sample collection,treatment and first challenge. Subsequently, they were sampled and dosedin their individual pens. Animals were offered approximately 5 kgalfalfa hay, 16 kg silage, 6 kg concentrate and 0.5 kg straw daily in atotal mixed ration offered in two feedings (60:40 concentrate:roughagediet). They were bedded on straw only during the pre-treatment period.Water was provided ad libitum. Animals were adapted to the lactatingration for at least 10 days before treatments were administered. From agroup of 6 dry cows and 5 non-lactating heifers, ten were selected basedon previous exposure to challenge and general health. Animals werepaired by body weight and were randomly assigned to control and acarbosetreatment groups within pair, ensuring similar distribution of heifersand cows. On days 0-6 of treatment, animals received 1.07 mg acarbose/kgdissolved in ˜200 mL water through the rumen cannula just before AM(07:30) and PM (16:00) feedings. Treatment 1 animals received anequivalent amount of water only. On days 7 and 8, each animal wasadministered a challenge through the cannula. When pH reached ˜4.5 andthere was evidence of L-lactate production, acute acidosis wasconsidered to be induced. When an animal experienced acute acidosis bythese criteria, rumen contents were removed and the rumen inoculatedwith rumen contents from a donor animal. Animals were weighed on days—1and 5 for calculation of acarbose dosing and challenge amounts. Tomeasure rumen fluid pH animals were fitted with a harness to hold anautomatic pH data recording system. The rumen pH was recorded every 10min during the days of challenge until an animal experienced acuteacidosis. Rumen fluid samples were taken (˜50 mL) from the rumen cannulathrough a filtered sampling tube. Sampling times were just before eachchallenge, and 3, 6, 8, 10 and 12 hrs after each challenge. The pH wasmeasured immediately. Samples for VFA and lactate analysis were preparedby adding 10 ml of rumen fluid to 1 ml of a solution containing amixture of 25 g/100 ml metaphosphoric acid and 1.2 g/100 ml crotonicacid straight after collection. In some cases the sample was filteredthrough gauze before pH measurement and acid treatment. This mixture wascentrifuged for 10 min at 12000 g. One aliquot of the supernatant wasremoved for immediate lactate analysis, one frozen for subsequentlactate analysis using Sigma kit 826. A third aliquot was transferred toan auto-sampler vial for subsequent VFA analysis. An initialdetermination of L-lactate was made during the study to establishacidotic status using Sigma kit 735. Subsequently D and L-lactate werere-measured for statistical analysis as described below. VFA's weremeasured on a Hewlett Packard 6890 series gas chromatograph fitted withan auto-sampler and flame ionisation detector. The acids were resolvedon a SGE Ltd. 25 meter BP21 column (0.33 mm O.D., 0.22 mm I.D. 0.25 umfilm thickness). Nitrogen was used as the carrier gas with a flow rateof 1.9 ml/min. The oven temperature was 165° C., and injection ports anddetectors were held at 250° C. Concentrations were calculated by usingcrotonic acid as an internal standard, and the system was calibratedusing a standard solution containing acetic, propionic, butyric,iso-valeric and n-valeric acids.

Results:

pH: For the challenge pH data are presented as a calculation of the timethat rumen pH was below a range of cut-offs. The first challenge did notinduce acute acidosis, but after the second challenge, four of thecontrol cattle had rumen pH values below 4.5. The short duration belowpH 4.5 was due to their removal from the study at that point. Rumen pHremained above 5.0 in all the treated cattle, indicating that acarboseprevented acute acidosis following carbohydrate challenge. The treatmenteffect of reducing the number of cows that became acidotic was shown tobe significant (P<0.5) by a simple contingency table analysis.

Lactates: There was no lactate detected after the first challenge, butlevels increased to >50 mM for four of five controls within 10 hrs ofthe challenge on the second and remained at zero in all theacarbose-treated animals. The fifth control, which had pH of ˜5.0, had amaximum level of lactate of 6 mM. Mean D-, L- and total lactates fromthe second challenge are summarized in the table. All lactates werehigher in the control than treatment group and there was a trend fortreatment by time interaction (the differences became greater overtime).

Group Mean D-Lactate, L-lactate and Total Lactates in Samples from Day 2Challenges (mM) D-Lactate L-Lactate Total-Lactate Hours Post SecondAcar- Acar- Acar- Challenge Control bose Control bose Control bose 011.48 0.67 3.82 0.35 15.30 1.02 3 42.93 3.63 15.33 1.28 58.26 4.91 558.19 10.91 17.80 3.08 76.08 13.99 7 74.69 18.00 18.98 4.93 93.92 22.93P Values Treatment .04 .03 .04 Time <.01 <.01 <.01 Treatment .13 .14 .12*Time

DISCUSSION/CONCLUSIONS

Twice a day acarbose treatment reduced pH responses to a highcarbohydrate load and blocked L-lactate production in response to theload. The pH responses to the first challenge were similar for the twogroups; it was the second challenge that allowed distinction. This issimilar to the observations of Cowe, et al. (J. Anim. Sci. 77:2259,1999) in which acute acidosis is induced after multiple challenges.

1. A formulation, which comprises:

in admixture with a suitable excipient, diluent or carrier, and anoptional stabilizer, selected with regard to the intended route ofadministration and standard pharmaceutical/veterinary/farming practice.2. A compound of the formula:

or veterinarily acceptable salt, solvate, hydrate or prodrug thereof.